Application of a Novel Method for the Comparison of DNA Binding Parameters of the Two Human Thyroid Hormone Receptor Subtypes hTRα1 and hTRβ1

Abstract

DNA-binding characteristics of the two human thyroid hormone receptors α₁ and β₁ (hTRα₁ and hTRβ₁) were studied by applying the recently developed solid-phase scintillation technique. Biotinylated double stranded oligonucleotides containing thyroid hormone response elements (TRE) were immobilized to streptavidin coated scintillating microtiter plates. The TRE:s consisted of variants of the consensus core sequence AGGTCA as monomers or as dimers in direct repeats. Equilibrium binding of radioactive labelled hTRα₁ and hTRβ₁ were studied. Metabolically ³⁵S-labelled hTR (in vitro translated cDNA) as well as hTR expressed in the baculovirus-system and labelled with ¹²⁵I-triiodothyronine (¹²⁵I-T₃) were used. In binding saturation experiments, the affinity for the TRE:s investigated did not differ greatly between hTRα₁ and hTRβ₁. No significant effects of T₃ on the amplitude of DNA binding of either hTRα₁ or hTRβ₁ to the single site response elements could be demonstrated. Receptor binding to direct repeats was stimulated by the hormone in the case of the hTRβ₁. The hTRα₁ binding to direct repeats was not significantly altered by T₃. The single site octameric variant of a TRE. TAAGGTCA, was observed to bind tighter to the hTR:s as compared to the hexameric variant AGGTCA. In the binding competition format, with one response element immobilized and other (un-biotinylated) added to the reaction mixture, there was a larger dymanic range for the affinity constants (IC50) as compared to the affinity constants (K_d) obtained in the binding saturation experiments. The present quantitative results confirm previous reports obtained with qualitative methods like gel shift assays. The method described here is applicable in basic research concerning characterisation of DNA binding of nuclear receptors. It also lends itself to automatization in high capacity formats.     

Binding between Prion Protein and Aβ Oligomers Contributes to the Pathogenesis of Alzheimer's Disease

A plethora of evidence suggests that protein misfolding and aggregation are underlying mechanisms of various neurodegenerative diseases, such as prion diseases and Alzheimer's disease(AD). Like prion diseases, AD has been considered as an infectious disease in the past decades as it shows strain specificity and transmission potential. Although it remains elusive how protein aggregation leads to AD, it is becoming clear that cellular prion protein(PrP~C ) plays an important role in AD pathogenesis. Here, we briefly reviewed AD pathogenesis and focused on recent progresses how PrP~C contributed to AD development. In addition, we proposed a potential mechanism to explain why infectious agents, such as viruses, conduce AD pathogenesis. Microbe infections cause Aβ deposition and upregulation of PrP~C , which lead to high affinity binding between Aβ oligomers and PrP~C . The interaction between PrP~C and Aβ oligomers in turn activates the Fyn signaling cascade, resulting in neuron death in the central nervous system(CNS). Thus, silencing PrP~C expression may turn out be an effective treatment for PrP~C dependent AD.     

Demonstration of a Direct Interaction between β2-Adrenergic Receptor and Insulin Receptor by BRET and Bioinformatics

Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and β₂-adrenergic receptor (β₂AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on β₂AR properties, we showed that IR coexpression had no effect on the ligand binding properties of β₂AR; however, IR reduced β₂AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between β₂AR and IR, we employed quantitative BRET² saturation and competition assays. Saturation assay data suggested constitutive β₂AR and IR homo- and heteromerization. Calculated acceptor/donor (AD₅₀) values as a measure of the relative affinity for homo- and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET² signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET² data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of β₂AR and IR identified intracellular domains encompassing residues at the end of the 7^th TM domain and C-terminal tail of β₂AR and a cytoplasmic part of the IR β chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order β₂AR:IR oligomer formation, likely comprising heteromers of homodimers.     

High Affinity of Interaction between Superantigen and T Cell Receptor Vβ Molecules Induces a High Level and Prolonged Expansion of Superantigen-reactive CD4+ T Cells

In mice implanted with an osmotic pump filled with the superantigen (SAG) staphylococcal enterotoxin A (SEA), the Vβ3⁺CD4⁺ T cells exhibited a high level of expansion whereas the Vβ11⁺CD4⁺ T cells exhibited a mild level of expansion. In contrast, in mice implanted with an osmotic pump filled with SE-like type P (SElP, 78.1% homologous with SEA), the Vβ11⁺CD4⁺ T cells exhibited a high level of expansion while the Vβ3⁺CD4⁺ T cells exhibited a low level of expansion, suggesting that the level of the SAG-induced response is determined by the affinities between the TCR Vβ molecules and SAG. Analyses using several hybrids of SEA and SElP showed that residue 206 of SEA determines the response levels of Vβ3⁺CD4⁺ and Vβ11⁺CD4⁺ T cells both in vitro and in vivo. Analyses using the above-mentioned hybrids showed that the binding affinities between SEA and the Vβ3/Vβ11 β chains and between SEA-MHC class II-molecule complex and Vβ3⁺/Vβ11⁺ CD4⁺ T cells determines the response levels of the SAG-reactive T cells both in vitro and in vivo.     

The Asymmetric Binding of PGC-1α to the ERRα and ERRγ Nuclear Receptor Homodimers Involves a Similar Recognition Mechanism

Background

PGC-1α is a crucial regulator of cellular metabolism and energy homeostasis that functionally acts together with the estrogen-related receptors (ERRα and ERRγ) in the regulation of mitochondrial and metabolic gene networks. Dimerization of the ERRs is a pre-requisite for interactions with PGC-1α and other coactivators, eventually leading to transactivation. It was suggested recently (Devarakonda et al) that PGC-1α binds in a strikingly different manner to ERRγ ligand-binding domains (LBDs) compared to its mode of binding to ERRα and other nuclear receptors (NRs), where it interacts directly with the two ERRγ homodimer subunits.

Methods/Principal Findings

Here, we show that PGC-1α receptor interacting domain (RID) binds in an almost identical manner to ERRα and ERRγ homodimers. Microscale thermophoresis demonstrated that the interactions between PGC-1α RID and ERR LBDs involve a single receptor subunit through high-affinity, ERR-specific L3 and low-affinity L2 interactions. NMR studies further defined the limits of PGC-1α RID that interacts with ERRs. Consistent with these findings, the solution structures of PGC-1α/ERRα LBDs and PGC-1α/ERRγ LBDs complexes share an identical architecture with an asymmetric binding of PGC-1α to homodimeric ERR.

Conclusions/Significance

These studies provide the molecular determinants for the specificity of interactions between PGC-1α and the ERRs, whereby negative cooperativity prevails in the binding of the coactivators to these receptors. Our work indicates that allosteric regulation may be a general mechanism controlling the binding of the coactivators to homodimers.     

Mapping the Receptor Binding Domain of Interleukin-1 Beta by Means of Binding Studies Using Overlapping Sequence Fragments: Why did it Fail?

Abstract

Interleukin-1 (IL-1) alpha and beta are polypeptide hormones that mediate a broad range of biological activities and interact with surface receptors on numerous cell types. Great efforts are made at present to define the interaction domain of IL-1 with its receptor. We have tried to map the domain of IL-1 beta by assessing the receptor interaction of synthetic octapeptide acid amides representing overlapping segments of the IL-1 beta primary sequence. Since the tertiary structure of IL-1 beta is known, the selection of octapeptides could be confined to the surface exposed residues. More than a 100 octapeptides were tested for binding in a competitive binding assay, using a mouse thymoma cell line (EL 4.61) as a receptor source and ¹²⁵I-IL-1 alpha and beta as radioligands. No binding was found at up to a one hundred fold excess of octapeptide over radioligand. From this lack of binding we conclude that the entropic cost of conformationally freezing the octapeptide is high and that the conformation of the binding domain is per se in terms of free energy and is stabilized by the overall tertiary structure of IL-1.     

Splenic Leukocytes Traffic to the Thyroid and Produce a Novel TSHβ Isoform during Acute Listeria monocytogenes Infection in Mice

The thyroid stimulating hormone beta-subunit (TSHβ) with TSHα form a glycoprotein hormone that is produced by the anterior pituitary in the hypothalamus-pituitary-thyroid (HPT) axis. Although TSHβ has been known for many years to be made by cells of the immune system, the role of immune system TSH has remained unclear. Recent studies demonstrated that cells of the immune system produce a novel splice variant isoform of TSHβ (TSHβv), but little if any native TSHβ. Here, we show that within three days of systemic infection of mice with Listeria monocytogenes, splenic leukocytes synthesized elevated levels of TSHβv. This was accompanied by an influx of CD14⁺, Ly6C⁺, Ly6G⁺ cells into the thyroid of infected mice, and increased levels of intrathyroidal TSHβv gene expression. Adoptive transfer of carboxyfluorescein succinimidyl ester (CFSE)-labeled splenic leukocytes from infected mice into non-infected mice migrated into the thyroid as early as forty-eight hours post-cell transfer, whereas CFSE-labeled cells from non-infected mice failed to traffic to the thyroid. These findings demonstrate for the first time that during bacterial infection peripheral leukocytes produce elevated levels of TSHβv, and that spleen cells traffic to the thyroid where they produce TSHβv intrathyroidally.     

Binding of BIS like and other ligands with the GSK-3β kinase: a combined docking and MM-PBSA study

Binding of several bisindolylmaleimide (BIS) like (BIS-3, BIS-8 and UCN1) and other ligands (H89, SB203580 and Y27632) with the glycogen synthase kinase-3 (GSK-3β) has been studied using combined docking, molecular dynamics and Poisson-Boltzmann surface area analysis approaches. The study generated novel binding modes of these ligands that can rationalize why some ligands inhibit GSK-3β while others do not. The relative binding free energies associated with these binding modes are in agreement with the corresponding measured specificities. This study further provides useful insight regarding possible existence of multiple conformations of some ligands like H89 and BIS-8. It is also found that binding modes of BIS-3, BIS-8 and UCN1 with GSK-3β and PDK1 kinases are similar. These new insights are expected to be useful for future rational design of novel, more potent GSK-3β-specific inhibitors as promising therapeutics.     

Effects of β-Amyloid-(25-35) Peptides on Radioligand Binding to Excitatory Amino Acid Receptors and Voltage-Dependent Calcium Channels: Evidence for a Selective Affinity for the Glutamate and Glycine Recognition Sites of the NMDA Receptor

The neurotoxic fragment corresponding to residues 25-35 of the -amyloid (A) peptide [A-(25-35)] has been shown to exert effects on (+)-[³H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate ([³H]MK-801) binding to the cation channel of the N-methyl-D-aspartate (NMDA) receptor. In the present study, we investigated whether the amidated and carboxylic acid C-terminated forms of A-(25-35) [A-(25-35-NH₂) and A-(25-35-COOH), respectively] exert effects on other excitatory amino acid receptor and cation channel types in rat cortical membranes. Both A-(25-35-NH₂) and A-(25-35-COOH) gave statistically significant dose-dependent inhibitions of [³H]glutamate and [³H]glycine binding to the agonist recognition sites of the NMDA receptor. Ten M A-(25-35-NH₂) and A-(25-35-COOH) gave 25% and 20% inhibitions of [³H]glutamate binding and 75% and 70% inhibitions of [³H]glycine binding, respectively. A-(25-35-NH₂), but not A-(25-35-COOH), gave a small (ca. 17% at 10 M) statistically significant increase of [³H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([³H]AMPA) binding. [³H]kainate binding was not significantly affected by either peptide. Similarly, neither peptide affected either the maximal level or EC₅₀ value for calcium stimulation of [³H]nitrendipine binding. It is concluded that A-(25-35) shows slight affinity for the agonist recognition sites of the NMDA receptor, but not for other excitatory amino acid receptor types or for L-type voltage-dependent calcium channels.     

Meta-Analysis of the Association between COX-2 Polymorphisms and Risk of Colorectal Cancer Based on Case–Control Studies

Objective

Cyclooxygenase-2 (COX-2) is an inducible enzyme converting arachidonic acid to prostaglandins and playing important roles in inflammatory diseases as well as tumor development. Previous studies investigating the association between COX-2 polymorphisms and colorectal cancer (CRC) risk reported conflicting results. We performed a meta-analysis of all available studies to explore this association.

Methods

All studies published up to October 2013 on the association between COX-2 polymorphisms and CRC risk were identified by searching electronic databases PubMed, EMBASE, and Cochrane library. The association between COX-2 polymorphisms and CRC risk was assessed by odds ratios (ORs) together with their 95% confidence intervals (CIs).

Results

Ten studies with 6,774 cases and 9,772 controls were included for −1195A>G polymorphism, 13 studies including 6,807 cases and 10,052 controls were available for −765G>C polymorphism, and 8 studies containing 5,121 cases and 7,487 controls were included for 8473T>C polymorphism. With respect to −765G>C polymorphism, we did not find a significant association with CRC risk when all eligible studies were pooled into the meta-analysis. However, in subgroup analyses by ethnicity and cancer location, with a Bonferroni corrected alpha of 0.05/2, statistical significant increased CRC risk was found in the Asian populations (dominant model CC+CG vs. GG: OR = 1.399, 95%CI: 1.113–1.760, P = 0.004) and rectum cancer patients (CC vs. GG: OR = 2.270, 95%CI: 1.295–3.980, P = 0.004; Recessive model CC vs. CG+GG: OR = 2.269, 95%CI: 1.297–3.970, P = 0.004). In subgroup analysis according to source of control, no significant association was detected. With respect to −1195A>G and 8473T>C polymorphisms, no significant association with CRC risk was demonstrated in the overall and subgroup analyses.

Conclusions

The present meta-analysis suggests that the COX-2 −765G>C polymorphism may be a risk factor for CRC in Asians and rectum cancer patients. Further large and well-designed studies are needed to confirm this association.     

Both Functional LTβ Receptor and TNF Receptor 2 Are Required for the Development of Experimental Cerebral Malaria

Background

TNF-related lymphotoxin α (LTα) is essential for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The pathway involved has been attributed to TNFR2. Here we show a second arm of LTα-signaling essential for ECM development through LTβ-R, receptor of LTα1β2 heterotrimer.

Methodology/Principal Findings

LTβR deficient mice did not develop the neurological signs seen in PbA induced ECM but died at three weeks with high parasitaemia and severe anemia like LTαβ deficient mice. Resistance of LTαβ or LTβR deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and angiography, associated with lack of microvascular obstruction, while wild-type mice developed distinct microvascular pathology. Recruitment and activation of perforin⁺ CD8⁺ T cells, and their ICAM-1 expression were clearly attenuated in the brain of resistant mice. An essential contribution of LIGHT, another LTβR ligand, could be excluded, as LIGHT deficient mice rapidly succumbed to ECM.

Conclusions/Significance

LTβR expressed on radioresistant resident stromal, probably endothelial cells, rather than hematopoietic cells, are essential for the development of ECM, as assessed by hematopoietic reconstitution experiment. Therefore, the data suggest that both functional LTβR and TNFR2 signaling are required and non-redundant for the development of microvascular pathology resulting in fatal ECM.     

Amyloid-β Acts as a Regulator of Neurotransmitter Release Disrupting the Interaction between Synaptophysin and VAMP2

Background

It is becoming increasingly evident that deficits in the cortex and hippocampus at early stages of dementia in Alzheimer’s disease (AD) are associated with synaptic damage caused by oligomers of the toxic amyloid-β peptide (Aβ42). However, the underlying molecular and cellular mechanisms behind these deficits are not fully understood. Here we provide evidence of a mechanism by which Aβ42 affects synaptic transmission regulating neurotransmitter release.

Methodology/Findings

We first showed that application of 50 nM Aβ42 in cultured neurones is followed by its internalisation and translocation to synaptic contacts. Interestingly, our results demonstrate that with time, Aβ42 can be detected at the presynaptic terminals where it interacts with Synaptophysin. Furthermore, data from dissociated hippocampal neurons as well as biochemical data provide evidence that Aβ42 disrupts the complex formed between Synaptophysin and VAMP2 increasing the amount of primed vesicles and exocytosis. Finally, electrophysiology recordings in brain slices confirmed that Aβ42 affects baseline transmission.

Conclusions/Significance

Our observations provide a necessary and timely insight into cellular mechanisms that underlie the initial pathological events that lead to synaptic dysfunction in Alzheimer’s disease. Our results demonstrate a new mechanism by which Aβ42 affects synaptic activity.