A mean field model of ligand-protein interactions: implications for the structural assessment of human immunodeficiency virus type 1 protease complexes and receptor-specific binding.

We propose a general mean field model of ligand-protein interactions to determine the thermodynamic equilibrium of a system at finite temperature. The method is employed in structural assessments of two human immuno-deficiency virus type 1 protease complexes where the gross effects of protein flexibility are incorporated by utilizing a data base of crystal structures. Analysis of the energy spectra for these complexes has revealed that structural and thermo-dynamic aspects of molecular recognition can be rationalized on the basis of the extent of frustration in the binding energy landscape. In particular, the relationship between receptor-specific binding of these ligands to human immunodeficiency virus type 1 protease and a minimal frustration principle is analyzed.

A type 1 serine/threonine kinase receptor that can dorsalize mesoderm in Xenopus.

We have cloned a type I serine/threonine kinase receptor, XTrR-I, from Xenopus. XTrR-I (Xenopus transforming growth factor beta-related receptor type I) is expressed in all regions of embryos throughout early development. Overexpression of this receptor does not affect ectoderm or endoderm but dorsalizes the mesoderm such that muscle appears in ventral mesoderm and notochord appears in lateral mesoderm normally fated to become muscle. In addition, overexpression of XTrR-I in UV-treated embryos is able to cause formation of a partial dorsal axis. These results suggest that XTrR-I encodes a receptor which responds in normal development to a transforming growth factor beta-like ligand so as to promote dorsalization. Its function would therefore be to direct mesodermalized tissue into muscle or notochord.

A progesterone metabolite stimulates the release of gonadotropin-releasing hormone from GT1-1 hypothalamic neurons via the gamma-aminobutyric acid type A receptor.

The reduced progesterone metabolite tetrahydroprogesterone (3 alpha-hydroxy-5 alpha-pregnan-20-one; 3 alpha,5 alpha-THP) is a positive modulator of the gamma-aminobutyric acid type A (GABAA) receptor. Experiments performed in vitro with hypothalamic fragments have previously shown that GABA could modulate the release of gonadotropin-releasing hormone (GnRH). Using GT1-1 immortalized GnRH neurons, we investigated the role of GABAA receptor ligands, including 3 alpha,5 alpha-THP, on the release of GnRH. We first characterized the GABAA receptors expressed by these neurons. [3H]Muscimol, but not [3H]flunitrazepam, bound with high affinity to GT1-1 cell membranes (Kd = 10.9 +/- 0.3 nM; Bmax = 979 +/- 12 fmol/mg of protein), and [3H]muscimol binding was enhanced by 3 alpha,5 alpha-THP. mRNAs encoding the alpha 1 and beta 3 subunits of the GABAA receptor were detected by the reverse transcriptase polymerase chain reaction. In agreement with binding data, the benzodiazepine-binding gamma subunit mRNA was absent. GnRH release studies showed a dose-related stimulating action of muscimol. 3 alpha,5 alpha-THP not only modulated muscimol-induced secretion but also stimulated GnRH release when administered alone. Bicuculline and picrotoxin blocked the effects of 3 alpha,5 alpha-THP and muscimol. Finally, we observed that GT1-1 neurons convert progesterone to 3 alpha,5 alpha-THP. We propose that progesterone may increase the release of GnRH by a membrane mechanism, via its reduced metabolite 3 alpha,5 alpha-THP acting at the GABAA receptor.

Hodgkin's lymphoma cell lines express a fusion protein encoded by intergenically spliced mRNA for the multilectin receptor DEC-205 (CD205) and a novel C-type lectin receptor DCL-1

Classic Hodgkin's lymphoma (HL) tissue contains a small population of morphologically distinct malignant cells called Hodgkin and Reed-Sternberg (HRS) cells, associated with the development of HL. Using 3'-rapid amplification of cDNA ends ( RACE) we identified an alternative mRNA for the DEC-205 multilectin receptor in the HRS cell line L428. Sequence analysis revealed that the mRNA encodes a fusion protein between DEC-205 and a novel C-type lectin DCL-1. Although the 7.5-kb DEC-205 and 4.2-kb DCL-1 mRNA were expressed independently in myeloid and B lymphoid cell lines, the DEC-205/DCL-1 fusion mRNA (9.5 kb) predominated in the HRS cell lines ( L428, KM-H2, and HDLM-2). The DEC-205 and DCL-1 genes comprising 35 and 6 exons, respectively, are juxtaposed on chromosome band 2q24 and separated by only 5.4 kb. We determined the DCL-1 transcription initiation site within the intervening sequence by 5'-RACE, confirming that DCL-1 is an independent gene. Two DEC-205/DCL-1 fusion mRNA variants may result from cotranscription of DEC-205 and DCL-1, followed by splicing DEC-205 exon 35 or 34-35 along with DCL-1 exon 1. The resulting reading frames encode the DEC-205 ectodomain plus the DCL-1 ectodomain, the transmembrane, and the cytoplasmic domain. Using DCL-1 cytoplasmic domain-specific polyclonal and DEC-205 monoclonal antibodies for immunoprecipitation/Western blot analysis, we showed that the fusion mRNA is translated into a DEC-205/DCL-1 fusion protein, expressed in the HRS cell lines. These results imply an unusual transcriptional control mechanism in HRS cells, which cotranscribe an mRNA containing DEC-205 and DCL-1 prior to generating the intergenically spliced mRNA to produce a DEC-205/DCL-1 fusion protein.

Type 1 ryanodine receptor interactions

Excitation-contraction coupling is an essential part of skeletal muscle contraction. It encompasses the sensing of depolarisation of the plasma membrane coupled with the release of Ca2+ from intracellular stores. The channel responsible for this release is called the Ryanodine receptor (RyR), and forms a hub of interacting proteins which work in concert to regulate the release of Ca2+ through this channel. The aim of this work was to characterise possible novel interactions with a proline-rich region of the RyR1, to characterise a monoclonal antibody (mAb VF1c) raised against a junctional sarcoplasmic reticulum protein postulated to interact with RyR1, and to characterise the protein recognised by this antibody in models of skeletal muscle disease such as Duchenne Muscular dystrophy (DMD) and sarcopenia. These experiments were performed using cell culture, protein purification via immunoprecipitation, affinity purification, low pressure chromatography and western blotting techniques. It was found that the RyR1 complex isolated from rat skeletal muscle co-purifies with the Growth factor receptor bound protein 2 (GRB2), very possibly via an interaction between the proline rich region of RyR1 and one of the SH3 domains located on the GRB2 protein. It was also found that Pleiotrophin and Phospholipase Cγ1, suggested interactors of the proline rich region of RyR1, did not co-purify with the RyR1 complex. Characterisation of mAb VF1c determined that this monoclonal antibody interacts with junctophilin 1, and binds to this protein between the region of 369-460, as determined by western blotting of JPH1 fragments expressed in yeast. It was also found that JPH1 and JPH2 are differentially regulated in different muscles of rabbit, where the highest amount of both proteins was found in the extensor digitorum longus (EDL) muscle. JPH1 and 2 levels were also examined in three rodent models of disease: the mdx mouse (a model of DMD), chronic intermittent hypoxia (CIH)-treated rat, and aged and adult mice, a model of sarcopenia. In the EDL and soleus muscle of CIH treated rats, no difference in either JPH1 or JPH2 abundance was detected in either muscle. An examination of JPH1 and 2 expression in mdx and wild type controls diaphragm, vastus lateralis, soleus and gastrocnemius muscle found no major differences in JPH1 abundance, while JPH2 was decreased in mdx gastrocnemius compared to wild type. In a mouse model of sarcopenia, JPH1 abundance was found to be increased in aged soleus but not in aged quadriceps, while in exercised quadriceps, JPH2 abundance was decreased compared to unexercised controls. Taken together, these results have implications for the regulation of RyR1 and JPH1 and 2 in skeletal muscle in both physiological and pathological states, and provide a newly characterised antibody to expand the field of JPH1 research.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Functional implications of the human T-lymphotropic virus type 1 transmembrane glycoprotein helical hairpin structure

Retrovirus entry into cells follows receptor binding by the surface exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

Purpose We evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the antihypertensive drug losartan (LOS). Results We showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumorassociated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C. Conclusions Together, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti-B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.

Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

Angiotensin II (Ang II) and vascular endothelial growth factor (VEGF) are important mediators of kidney injury in diabetes. Acute hyperglycemia increased synthesis of intrarenal Ang I and Ang II and resulted in activation of both Ang II receptors, AT1 and AT2, in the kidney. Losartan (specific AT1 antagonist) or PD123319 (specific AT2 antagonist) did not affect hyperglycemia but prevented activation of renal AT1 and AT2, respectively. In murine renal cortex, acute hyperglycemia increased VEGF protein but not mRNA content after 24 h, which suggested translational regulation. Blockade of AT2, but not AT1, prevented increase in VEGF synthesis by inhibiting translation of VEGF mRNA in renal cortex. Acute hyperglycemia increased VEGF expression in wild type but not in AT2 knockout mice. Binding of heterogeneous nuclear ribonucleoprotein K to VEGF mRNA, which stimulates its translation, was prevented by blockade of AT2, but not AT1. The Akt-mTOR-p70(S6K) signaling pathway, involved in the activation of mRNA translation, was activated in hyperglycemic kidneys and was blocked by the AT2 antagonist. Elongation phase is an important step of mRNA translation that is controlled by elongation factor 1A (eEF1A) and 2 (eEF2). Expression of eEF1A and activity of eEF2 was higher in kidney cortex from hyperglycemic mice and only the AT2 antagonist prevented these changes. To assess selectivity of translational control of VEGF expression, we measured expression of fibronectin (FN) and laminin beta 1 (lam beta 1): acute hyperglycemia increased FN expression at both protein and mRNA levels, indicating transcriptional control, and did not affect the expression of lam beta 1. To confirm results obtained with PD123319, we induced hyperglycemia in AT2 knockout mice and found that in the absence of AT2, translational control of VEGF expression by hyperglycemia was abolished. Our data show that acute hyperglycemia stimulates Ang II synthesis in murine kidney cortex, this leads to AT2 activation and stimulation of VEGF mRNA translation, via the Akt-mTOR-p70(S6K) signaling pathway. Our data show that exclusive translational control of protein expression in the kidney by acute hyperglycemia is not a general phenomenon, but do not prove that it is restricted to VEGF. (C) 2010 Elsevier Inc. All rights reserved.

DC-SIGN (CD209) gene promoter polymorphisms in a Brazilian population and their association with human T-cell lymphotropic virus type 1 infection

This study evaluated four polymorphisms located in the DC-SIGN (CD209) gene promoter region (positions -336, -332 -201 and -139) in DNA samples from four Brazilian ethnic groups (Caucasians, Afro-Brazilian, Asians and Amerindians) to establish the population distribution of these single-nucleotide polymorphisms (SNPs) and correlated DC-SIGN polymorphisms and infection in samples from human T-cell lymphotropic virus type 1 (HTLV-1)-infected individuals. To identify CD209 SNPs, 452 bp of the CD209 promoter region were sequenced and the genotype and allelic frequencies were evaluated. This is the first study to show genetic polymorphism in the CD209 gene in distinct Brazilian ethnic groups with the distribution of allelic and genotypic frequency. The results showed that -336A and -139A SNPs were quite common in Asians and that the -201T allele was not observed in Caucasians, Asians or Amerindians. No significant differences were observed between individuals with HTLV-1 disease and asymptomatic patients. However, the -336A variant was more frequent in HTLV-1 -infected patients [HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), 80%; healthy asymptomatic HTLV-1 carriers, 90 %] than in the control group (70 %) [P=0.0197, odds ratio (OR)=2.511, 95 % confidence interval (CI)=1.218-5.179). In addition, the -139A allele was found to be associated with protection against HTLV-1 infection (P=0.0037, OR=0.3758, 95% CI=0.1954-0.7229) when the HTLV-1 -infected patients as a whole were compared with the healthy-control group. These observations suggest that the -139A allele may be associated with HTLV-1 infection, although no significant association was observed among asymptomatic and HAM/TSP patients. In conclusion, the variation observed in SNPs -336 and -139 indicates that this lectin may be of crucial importance in the susceptibility/transmission of HTLV-1 infections.

Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases

Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation. Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10). Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1 alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CK beta 8/CCL23, and osteoprotegerin, which were significantly higher than in control. Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

Cysteinyl-leukotriene type 1 receptors transduce a critical signal for the up-regulation of eosinophilopoiesis by interleukin-13 and eotaxin in murine bone marrow

IL-13 and eotaxin play important, inter-related roles in asthma models. In the lungs, CysLT, produced by the 5-LO-LTC4S pathway, mediate some local responses to IL-13 and eotaxin; in bone marrow, CysLT enhance IL-5-dependent eosinophil differentiation. We examined the effects of IL-13 and eotaxin on eosinophil differentiation. Semi-solid or liquid cultures were established from murine bone marrow with GM-CSF or IL-5, respectively, and the effects of IL-13, eotaxin, or CysLT on eosinophil colony formation and on eosinophil differentiation in liquid culture were evaluated, in the absence or presence of: a) the 5-LO inhibitor zileuton, the FLAP inhibitor MK886, or the CysLT1R antagonists, montelukast and MK571; b) mutations that inactivate 5-LO, LTC4S, or CysLT1R; and c) neutralizing mAb against eotaxin and its CCR3 receptor. Both cytokines enhanced GM-CSF-dependent eosinophil colony formation and IL-5-stimulated eosinophil differentiation. Although IL-13 did not induce eotaxin production, its effects were abolished by anti-eotaxin and anti-CCR3 antibodies, suggesting up-regulation by IL-13 of responses to endogenous eotaxin. Anti-CCR3 blocked eotaxin completely. The effects of both cytokines were prevented by zileuton, MK886, montelukast, and MK571, as well as by inactivation of the genes coding for 5-LO, LTC4S, and CysLT1R. In the absence of either cytokine, these treatments or mutations had no effect. These findings provide evidence for: a) a novel role of eotaxin and IL-13 in regulating eosinophilopoiesis; and b) a role for CysLTRs in bone marrow cells in transducing cytokine regulatory signals. J. Leukoc. Biol. 87: 885-893; 2010.

The therapeutic potential of endothelin-1 receptor antagonists and endothelin-converting enzyme inhibitors on the cardiovascular system

Clinical trials have established bosentan, an orally active non-selective endothelin (ET) receptor antagonist, as a beneficial treatment in pulmonary hypertension. Trials have also shown short-term benefits of bosentan in systemic hypertension and congestive heart failure. However, bosentan also increased plasma levels of ET-1, probably by inhibiting the clearance of ET-1 by endothelin type B (ET.) receptors, and this may mean its effectiveness is reduced with long-term clinical use. Preliminary data suggests that selective endothelin type A (ETA) receptor antagonists (BQ-123, sitaxsentan) may be more beneficial than the non-selective ET receptor antagonists in heart failure, especially when the failure is associated with pulmonary hypertension. Experimental evidence in animal disease models suggests that non-selective ET or selective ETA receptor antagonism may have a role in the treatment of athero-sclerosis, restenosis, myocarditis, shock and portal hypertension. In animal models of myocardial infarction and/or reperfusion injury, non-selective ET or selective ETA receptor antagonists have beneficial or detrimental effects depending on the conditions and agents used. Thus clinical trials of the nonselective ET or selective ETA receptor antagonists in these conditions are not presently warranted. Several selective endothelin-converting enzyme inhibitors tors have been synthesised recently, and these are only beginning to be tested in animal models of cardiovascular disease, and thus the clinical potential of these inhibitors is still to be defined.