CXCL-10/CXCR3 in macrophages regulates tissue repair by controlling the expression of Arg1, VEGFa and TNFα.

Macrophages have been reported to participate in inflammation, tissue homeostasis and tissue repair. The detailed mechanism of macrophage-mediated tissue repair is not clear. CXCL-10, secreted by monocytes, endothelial cells and fibroblasts, mediates immune response and angiogenesis by binding to CXCR3. In this study, the expression of CXCL-10 and CXCR3 in porcine lung injury induced by porcine reproductive and respiratory syndrome virus (PRRSV) infection was firstly examined. The results showed that the expression of both CXCL-10 and CXCR3 increased in the infected pig lungs. In addition, the increased expression of CXCL-10 and CXCR3 in macrophage treated by poly (I:C) was also observed, suggesting the autocrine system existed in macrophages. Furthermore, CXCL-10 treatment induced upregulation of Arg1 and VEGFa, and downregulation of TNFα in macrophage, and CXCR3 antagonist AMG487 treatment presented the contrary effects on the expression of Arg1, VEGFa, and TNFα. CXCL- 10-stimulated effects were dependent on PI3K/Akt signaling pathway. Wound-healing assay showed that CXCL-10 treatment macrophage conditioned medium promoted the healing process of endothelial cells. Our results suggested that CXCL-10/CXCR3 in macrophage may mediate tissue repair by regulating the macrophage expression of Arg1, VEGFa and TNFα. Modulation of CXCL-10/CXCR3 axis in macrophage may be a potential therapeutic strategy for tissue injury and repair.

Transforming growth factor beta-1 expression in macrophages of human chronic periapical diseases.

The objective of this study was to observe the distribution of macrophages (MPs) expressing transforming growth factor beta-1 (TGF-ß1) in tissue samples from patients with different human chronic periapical diseases. In this study, samples were collected from 75 volunteers, who were divided into three groups according to classified standards, namely, healthy control (N = 25), periapical granuloma (N = 25), and periapical cyst (N = 25). The samples were fixed in 10% buffered formalin for more than 48 h, dehydrated, embedded, and stained with hematoxylin and eosin for histopathology. Double immunofluorescence was conducted to analyze the expression of TGF-ß-CD14 double-positive MPs in periapical tissues. The number of double-positive cells (cells/mm2) were significantly higher in the chronic periapical disease tissues (P < 0.01) compared to that in the control tissue; in addition, the density of TGF-ß1-CD14 double positive cells was significantly higher in the periapical cyst group than in the periapical granuloma group (P < 0.01). The number of TGF-ß1 expressing macrophages varied with human chronic periapical diseases. The TGF-ß1-CD14 double-positive cells might play an important role in the pathology of human chronic periapical diseases.

Expression of the stem cell factor in fibroblasts, endothelial cells, and macrophages in periapical tissues in human chronic periapical diseases.

Stem cell factor (SCF), an important stem cell cytokine, has multiple functions. Fibroblasts (FBs), mature mast cells, endothelial cells (ECs), and eosinophil granulocytes can produce SCF in the inflammatory process. Therefore, we aimed to observe SCF expression in FBs, ECs, and macrophages (MPs) in periapical tissues in human chronic periapical disease and investigate the effects of cells expressing SCF in pathogenesis of the disease. Healthy (N = 20), periapical cyst (N = 15), and periapical granuloma (N = 15) tissues were fixed in 10% formalin for 48 h, embedded in paraffin, and stained with hematoxylin and eosin to observe histological changes. SCF expression was observed in FBs, ECs, and MPs in periapical tissues by double immunofluorescence. CD334, CD31, and CD14 are specific markers of FBs, ECs, and MPs, respectively. Results showed that densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs were significantly increased in periapical tissue groups (P < 0.01). There were no significant differences in CD334-SCF double-positive FB and CD31-SCF double-positive EC levels between the two periapical tissue groups (P > 0.05). CD14-SCF double-positive MP density was considerably higher in periapical granulomas than in cysts (P < 0.01). FB, EC, and MP levels were significantly high and densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs improved considerably in chronic periapical tissues, suggesting that the cells might be related to occurrence, development, and pathogenesis of chronic periapical disease.

Galectins expressed differently in genetically susceptible C57BL/6 and resistant BALB/c mice during acute ocular Toxoplasma gondii infection.

Ocular toxoplasmosis (OT) caused by Toxoplasma gondii is a major cause of infectious uveitis, however little is known about its immunopathological mechanism. Susceptible C57BL/6 (B6) and resistant BALB/c mice were intravitreally infected with 500 tachyzoites of the RH strain of T. gondii. B6 mice showed more severe ocular pathology and higher parasite loads in the eyes. The levels of galectin (Gal)-9 and its receptors (Tim-3 and CD137), interferon (IFN)-γ, IL-6 and IL-10 were significantly higher in the eyes of B6 mice than those of BALB/c mice; however, the levels of IFN-α and -ß were significantly decreased in the eyes and CLNs of B6 mice but significantly increased in BALB/c mice after infection. After blockage of galectin-receptor interactions by α-lactose, neither ocular immunopathology nor parasite loads were different from those of infected BALB/c mice without α-lactose treatment. Although the expressions of Gal-9/receptor were significantly increased in B6 mice and Gal-1 and -3 were upregulated in both strains of mice upon ocular T. gondii infection, blockage of galectins did not change the ocular pathogenesis of genetic resistant BALB/c mice. However, IFN-α and -ß were differently expressed in B6 and BALB/c mice, suggesting that type I IFNs may play a protective role in experimental OT.

Effect of microRNA-21 on the proliferation of human degenerated nucleus pulposus by targeting programmed cell death 4.

This study aims to explore the effect of microRNA-21 (miR-21) on the proliferation of human degenerated nucleus pulposus (NP) by targeting programmed cell death 4 (PDCD4) tumor suppressor. NP tissues were collected from 20 intervertebral disc degeneration (IDD) patients, and from 5 patients with traumatic spine fracture. MiR-21 expressions were tested. NP cells from IDD patients were collected and divided into blank control group, negative control group (transfected with miR-21 negative sequences), miR-21 inhibitor group (transfected with miR-21 inhibitors), miR-21 mimics group (transfected with miR-21 mimics) and PDCD4 siRNA group (transfected with PDCD4 siRNAs). Cell growth was estimated by Cell Counting Kit-8; PDCD4, MMP-2,MMP-9 mRNA expressions were evaluated by qRT-PCR; PDCD4, c-Jun and p-c-Jun expressions were tested using western blot. In IDD patients, the expressions of miR-21 and PDCD4 mRNA were respectively elevated and decreased (both P<0.05). The miR-21 expressions were positively correlated with Pfirrmann grades, but negatively correlated with PDCD4 mRNA (both P<0.001). In miR-21 inhibitor group, cell growth, MMP-2 and MMP-9 mRNA expressions, and p-c-Jun protein expressions were significantly lower, while PDCD4 mRNA and protein expressions were higher than the other groups (all P<0.05). These expressions in the PDCD4 siRNA and miR-21 mimics groups was inverted compared to that in the miR-21 inhibitor group (all P<0.05). MiR-21 could promote the proliferation of human degenerated NP cells by targeting PDCD4, increasing phosphorylation of c-Jun protein, and activating AP-1-dependent transcription of MMPs, indicating that miR-21 may be a crucial biomarker in the pathogenesis of IDD.

Effect of microRNA-21 on the proliferation of human degenerated nucleus pulposus by targeting programmed cell death 4

This study aims to explore the effect of microRNA-21 (miR-21) on the proliferation of human degenerated nucleus pulposus (NP) by targeting programmed cell death 4 (PDCD4) tumor suppressor. NP tissues were collected from 20 intervertebral disc degeneration (IDD) patients, and from 5 patients with traumatic spine fracture. MiR-21 expressions were tested. NP cells from IDD patients were collected and divided into blank control group, negative control group (transfected with miR-21 negative sequences), miR-21 inhibitor group (transfected with miR-21 inhibitors), miR-21 mimics group (transfected with miR-21 mimics) and PDCD4 siRNA group (transfected with PDCD4 siRNAs). Cell growth was estimated by Cell Counting Kit-8; PDCD4, MMP-2,MMP-9 mRNA expressions were evaluated by qRT-PCR; PDCD4, c-Jun and p-c-Jun expressions were tested using western blot. In IDD patients, the expressions of miR-21 and PDCD4 mRNA were respectively elevated and decreased (both P<0.05). The miR-21 expressions were positively correlated with Pfirrmann grades, but negatively correlated with PDCD4 mRNA (both P<0.001). In miR-21 inhibitor group, cell growth, MMP-2 and MMP-9 mRNA expressions, and p-c-Jun protein expressions were significantly lower, while PDCD4 mRNA and protein expressions were higher than the other groups (all P<0.05). These expressions in the PDCD4 siRNA and miR-21 mimics groups was inverted compared to that in the miR-21 inhibitor group (all P<0.05). MiR-21 could promote the proliferation of human degenerated NP cells by targeting PDCD4, increasing phosphorylation of c-Jun protein, and activating AP-1-dependent transcription of MMPs, indicating that miR-21 may be a crucial biomarker in the pathogenesis of IDD.

Obvious emphysema on computed tomography during an acute exacerbation of chronic obstructive pulmonary disease predicts a poor prognosis.

BACKGROUND: Emphysematous change on computed tomography (CT) during the stable phase of chronic obstructive pulmonary disease (COPD) is reported to correlate with COPD prognosis. Acute exacerbation of COPD (AECOPD) is associated with a high risk of mortality and a poor prognosis. AIMS: This study aims to study the relationship between prognosis and emphysematous changes on CT during an AECOPD. METHODS: Histories were recorded, and CT acquired for 106 patients who visited the emergency department for an AECOPD. Emphysematous change was quantified by measuring the percentage of low-attenuation areas (LAA%) in the entire lung on CT images with a threshold of -950 Hounsfield units. Other factors that could influence AECOPD prognosis were also recorded on admission and analysed. At follow ups conducted in 1 year, patient survival, the modified Medical Research Council (mMRC) Dyspnoea Scale, and performance status (PS) were evaluated, and a COPD Assessment Test (CAT) was completed. RESULTS: The 1-year follow up was completed by 103 of 106 patients. The median LAA% was significantly higher in non-survivors (11%, n = 16) than in survivors (5.69%, n = 87) (P = 0.006) at the 1-year follow up. LAA% was significantly correlated with mMRC grade (r = 0.285, P = 0.008), PS (r = 0.397, P < 0.001) and CAT score (r = 0.27, P = 0.017) at the 3-month follow up, and with mMRC grade (r = 0.405, P < 0.001) and PS (r = 0.377, P < 0.001) at the 1-year follow up. LAA% > 7.5% was a significant predictor of 1-year mortality, higher mMRC and PS at the 3-month and 1-year follow ups, after adjustment for other prognostic predictors. CONCLUSION: Obvious emphysematous changes on CT (LAA% > 7.5%) during an AECOPD predicts a poor prognosis independent of other known indicators.

Community-acquired pneumonia in Shanghai, China: microbial etiology and implications for empirical therapy in a prospective study of 389 patients.

The aim of this multicenter study was to identify the causative pathogens of community-acquired pneumonia (CAP) in Shanghai, China, and to determine their susceptibility to antimicrobial agents. Pathogens obtained from 389 patients with documented CAP during 2001-2003 were identified by multiple diagnostic tools that included bacterial culture, polymerase chain reaction (PCR), and specific immunological assays. Susceptibility of the bacterial isolates was tested by the broth microdilution method. A specific pathogen was identified in 39.8% (155/389) of the patients: Haemophilus influenzae (n=80), Klebsiella spp. (n=15), Streptococcus pneumoniae (n=12), Staphylococcus aureus (n=6), Moraxella catarrhalis (n=1), other gram-negative organisms (n=9), and atypical pathogens that comprised Mycoplasma pneumoniae (n=42), Chlamydia pneumoniae (n=17), and Legionella pneumophila (n=2). Most H. influenzae isolates were susceptible to ampicillin (88.3%), and all were susceptible to macrolides. Of the S. pneumoniae isolates, 75% (9/12) were susceptible to penicillin, while 25% (3/12) were intermediately susceptible. H. influenzae and atypical pathogens are among the most important pathogens of CAP. Ampicillin, cephalosporins, and the newer fluoroquinolones can be used as empirical therapy for CAP in the Shanghai area. The efficacy of monotherapy with newer macrolides for CAP caused by S. pneumoniae requires further evaluation.

Chimers of two fused ADP/ATP carrier monomers indicate a single channel for ADP/ATP transport.

The mitochondrial ADP/ATP carrier (AAC) is generally believed to function as a homodimer (Wt. Wt). It remains unknown whether the two monomers possess two independent but fully anticooperative channels or they form a single central channel for nucleotide transport. Here we generated fusion proteins consisting of two tandem covalent-linked AAC monomers and studied the kinetics of ADP/ATP transport in reconstituted proteoliposomes. Functional 64-kDa fusion proteins Wt-Wt and Wt-R294A (wild-type AAC linked to a mutant having low ATP transport activity) were expressed in mitochondria of yeast transformants. Compared to homodimer Wt. Wt, the fusion protein Wt-Wt retained the transport activity and selectivity of ADP versus ATP. The strongly divergent selectivities of Wt and R294A were partially propagated in the Wt-R294A fusion protein, suggesting a limited cooperativity during solute translocation. The rates of ADP or ATP transport were significantly higher than those predicted by the two-channel model. Fusion proteins for Wt-R204L (Wt linked to an inactive mutant) and R204L-Wt were not expressed in aerobically grown yeast cells, which contained plasmid rearrangements that regenerated the fully active 32-kDa homodimer Wt. Wt, suggesting that these fusion proteins are inactive in ADP/ATP transport. These results favor a single binding center gated pore model [Klingenberg, M. (1991) in A Study of Enzymes, Vol. 2: pp. 367-388] in which two AAC subunits cooperate for a coordinated ADP/ATP exchange through a single channel.

Identification and characterization of a melanin-concentrating hormone receptor.

Melanin-concentrating hormone (MCH), a neuropeptide expressed in central and peripheral nervous systems, plays an important role in the control of feeding behaviors and energy metabolism. An orphan G protein-coupled receptor (SLC-1/GPR24) has recently been identified as a receptor for MCH (MCHR1). We report here the identification and characterization of a G protein-coupled receptor as the MCH receptor subtype 2 (MCHR2). MCHR2 has higher protein sequence homology to MCHR1 than any other G protein-coupled receptor. The expression of MCHR2 has been detected in many regions of the brain. In contrast to MCHR1, which is intronless in the coding region and is located at the chromosomal locus 22q13.3, the MCHR2 gene has multiple exons and is mapped to locus 6q21. MCHR2 is specifically activated by nanomolar concentrations of MCH, binds to MCH with high affinity, and signals through Gq protein. This discovery is important for a full understanding of MCH biology and the development of potential therapeutics for diseases involving MCH, including obesity.

A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells.

A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3'-dipropylthiadicarbocyanine iodide (DiSC(3)(5); DiSC(3)). In permeabilized cells, DiSC(3) fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K(+) and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H(+) transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential.

Structure-function relationship in UCP1.

The function of uncoupling protein (UCP1) as a H+ transporter regulated by nucleotide binding is elucidated. H+ transport requires fatty acids (FA) with relatively wide structural tolerance. The nucleotide binding site is specific for purine nucleotides and tolerates a number of derivatives. The strong pH dependency facilitates regulation of nucleotide binding and thus H+ translocation. The structure-function relationship of UCP1 has been analysed by various probes and by mutagenesis. According to our model, FA are a cofactor in H+ transport, providing H+ shuttling carboxyl groups in the translocation channel. By mutagenesis, additional H+ translocating groups at both sides of the translocation channel were found. Two pH sensors, controlling nucleotide binding, were identified in accordance with earlier postulates deduced from the pH dependence of nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP). A common pH sensor E190 and a specific pH sensor H214 for triphosphates only, control access to the phosphate binding moiety. The three mitochondrial carrier family characteristic intrahelical arginines are essential for nucleotide binding. Mutagenesis of other charged residues reveals their role in structure stabilisation and/or has more generalised effects due to charge relay networks in UCP1.

Stoichiometry and mechanism of assembly of SV40 T antigen complexes with the viral origin of DNA replication and DNA polymerase alpha-primase.

The interactions of simian virus 40 (SV40) large T antigen with DNA carrying the viral origin of DNA replication, as well as its interactions with cellular replication proteins, have been investigated by using fluorescent ATP analogues as specific probes. The enhanced fluorescence of 3'(2')-O-(2,4, 6-trinitrophenyl)adenosine diphosphate (TNP-ADP) induced by T antigen binding to the nucleotide was decreased upon binding of T antigen to origin DNA. Similarly, the enhanced fluorescence induced by T antigen binding to TNP-ADP or TNP-ATP was decreased upon binding to human DNA polymerase alpha-primase (pol alpha), but not to replication protein A (RPA). Fluorescence titrations revealed noncompetitive inhibition of TNP-ADP binding by origin DNA, and noncompetitive inhibition of TNP-ADP and TNP-ATP binding by pol alpha, suggesting that T antigen complexed with either origin DNA or pol alpha was not able to bind the TNP nucleotide. From these titrations, we have measured a binding stoichiometry of 11.5 +/- 0.8 T antigen monomers per viral origin DNA, in agreement with the double hexamer assembly of T antigen on the origin as reported earlier. The stoichiometry of pol alpha binding to T antigen was measured to be 5.5 +/- 0.6 mol of T antigen per mole of pol alpha. While monomeric T antigen-nucleotide complex was a preferred ligand over free T antigen in the double hexamer assembly reaction, preformed T antigen hexamers were incapable of forming double hexamers on the DNA. The results support a model in which double hexamer assembly on the viral origin occurs by successive binding of 12 free T antigen or monomeric T-nucleotide complexes to the DNA. In contrast with this stepwise assembly of T antigen monomers on DNA, hexameric T antigen was able to bind directly to pol alpha with concomitant release of the bound TNP nucleotide. The possible implications of these results for the mechanism of initiation of SV40 DNA replication are discussed.

Characterization of the nucleotide binding properties of SV40 T antigen using fluorescent 3'(2')-O-(2,4,6-trinitrophenyl)adenine nucleotide analogues.

ATP binding to the large tumor (T) antigen encoded by the simian virus 40 (SV40) genome plays an essential role in the replication of viral DNA [Fanning, E., and Knippers, R. (1992) Annu. Rev. Biochem. 61, 55-85]. To better explore the functions of T antigen during the replication process, we have studied the interactions of T antigen with fluorescent 3'(2')-O-(2,4,6-trinitrophenyl) (TNP) adenine nucleotide analogues. Binding of TNP-ATP and TNP-ADP was accompanied by an 8-fold fluorescence enhancement and a concomitant blue shift (11 nm) of the maximal emission wavelength; the intrinsic protein tryptophan fluorescence was quenched maximally by 50%. Both signals were utilized to characterize the nucleotide binding activity of T antigen. TNP-ATP and TNP-ADP bound to the ATP binding site with dissociation constants of 0.35 microM and 2.6 microM. TNP substitution enhanced the affinity of ADP for T antigen by approximately 11-fold. The binding stoichiometry was 1 mol of TNP nucleotide per mole of monomer T antigen. The binding of TNP-ATP was more temperature dependent than that of TNP-ADP. The enthalpy change contributed nearly half of the energy for TNP-ATP binding, whereas binding of TNP-ADP was primarily entropy driven. Both TNP-ATP and TNP-ADP were strong inhibitors of the T antigen ATPase activity, confirming the high affinities of the TNP nucleotides for the ATP binding site. Like the parent nucleotides, they also induced T antigen hexamer formation. Using the TNP nucleotides as fluorescent probes, we have measured the affinity of various nucleotides and analogues for T antigen. The results indicate that the nucleotide binding specificity of T antigen was similar to that of the prokaryotic helicases Dna B and Rep, suggesting closely related ATP binding sites in the three DNA helicases.

Slow-phase kinetics of nucleotide binding to the uncoupling protein from brown adipose tissue mitochondria.

The kinetics of nucleotide binding to the uncoupling protein (UCP) from brown adipose tissue mitochondria were studied with a filter binding method. Fast and slow phases of binding were observed, corresponding to the two-stage binding model based on equilibrium binding studies (Huang, S. G., and Klingenberg, M. (1996) Biochemistry 35, 7846-7854) (Reaction 1). [reaction: see text] Although this method determines total binding, only the slow phase can be resolved. The fast unresolved phase represents the formation of the initial loose UCP-nucleotide complex (UN; Kd approximately 2 microM), whereas the slow phase reflects the tight binding (U*N) associated with a conformational change induced by the bound nucleotide. Best fits of the binding data yielded, for the slow phase, k+1 values of 3.0 x 10(-3) s-1 for GTP, 4.8 x 10(-3) s-1 for ATP, 0.13 s-1 for GDP, and >0.7 s-1 for ADP and dissociation rate constants (k-1) of 0.10 x 10(-3) s-1 for GTP, 0.58 x 10(-3) s-1 for ATP, 8.8 x 10(-3) s-1 for GDP, and >0.3 s-1 for ADP at pH 6.7 and 4 degrees C. The rates were fairly pH- and temperature-dependent. The distribution constant Kc' (=k+1/k-1) between the tight and loose complexes ranged between 2 and 30, suggesting formation of 71-97% of the tight complex at equilibrium. The Kc' decreases with increasing pH, indicating a progressively less tight complex population. Anions (SO42-) form a loose complex with UCP, thus affecting the initial association step, but not the subsequent transition step. While the kinetic constants were verified by dilution and chase experiments as well as in mass action plots, they were further corroborated with data obtained by fluorescence competition measurements. Taken together, our results show that nucleotide binding to UCP occurs via a two-stage mechanism in which the initial loose complex rearranges slowly into a tight complex.

Chloride channel properties of the uncoupling protein from brown adipose tissue mitochondria: a patch-clamp study.

The uncoupling protein (UCP) from brown adipose tissue mitochondria possesses H+ and Cl- transport activities [reviewed in Klingenberg, M. (1990) Trends Biochem. Sci. 15, 108-112]. Being a member of a mitochondrial carrier family, the transport of H+ and Cl- is carrier-like, i.e., much slower as compared to channels. Here we report that UCP reconstituted into giant liposomes displays stable chloride channel properties under patch-clamp conditions. The transport inhibitors (GTP, GDP, ATP, and ADP) also inhibit this channel in a reversible way, showing that the channel activity is associated with UCP. The slightly inward-rectifying chloride channel has a unit conductance of approximately 75 pS in symmetrical 100 mM KCl and closes at high positive potentials on the matrix side of UCP. Channel gatings switch from slow open-closure transitions to fast flickerings as the holding potential increases over +60 mV. Substitution experiments reveal a strong discrimination against cations [P(Cl-)/P(K+) approximately 17] and a permeability ratio order of Cl- > Br- > F- > SCN- > I- > NO3- > SO4(2-) > HPO4(2-) > gluconate. Nucleotide inhibition studies indicate that 70% UCP molecules had its matrix side oriented outside in the giant liposomes. Fatty acids, pH, divalent cations (Ca2+ and Mg2+), and mersalyl do not influence these Cl- currents. The Cl- channel can be blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) from the matrix side of UCP. The data are consistent with a dimer consisting of two monomeric 75-pS Cl- channels or with a monomeric 150-pS channel having a 50% subconductance state. The channel current increases with Cl- concentration showing a typical saturation curve with Km approximately 63 mM and gmax approximately 120 pS (100 mM KCl in the pipet). The Cl- conductance measured under these conditions is 6 orders of magnitude higher than the Cl- transport activity reported earlier, suggesting that the UCP has the potential of behaving as an anion channel.

Two-stage nucleotide binding mechanism and its implications to H+ transport inhibition of the uncoupling protein from brown adipose tissue mitochondria.

The uncoupling protein (UCP) from brown adipose tissue mitochondria is the simplest H+ translocator known. H+ transport is regulated by fatty acids as activators and by pruine nucleotides as inhibitors. Nucleotide binding again is strongly influenced by the pH [Klingenberg, M. (1988) Biochemistry 27, 781-791]. Previously, by using fluorescent 2'-O-dansyl (DANS) derivatives of purine nucleotides, a two-stage binding mechanism was unraveled with a slow transition from a loose into a tight conformational state in the isolated UCP [Huang, S.-G., & Klingenberg, M. (1995) Biochemistry 34, 349-360]. Whereas with the unsubstituted nucleotides the transition to the tight state is nearly complete, various DANS and DAN (dimethylaminonaphthoyl) nucleotides bind more to the loose state. Here we investigated the relationships between the two-stage nucleotide binding and the inhibition of the H+ transport activity in reconstituted proteoliposomes. Further, limited tryptic digestion was used as an indicator of conformational change induced by the nucleotide binding in the isolated protein. The inhibition of H+ transport activity in reconstituted UCP proteoliposomes correlated only with the fraction of tight state of nucleotide binding. Unsubstituted nucleotides (ATP, GTP, and ADP) as well as DANSGTP inhibit fully the H+ transport, whereas DANSATP and DANSADP inhibit only to about 50%, and DANSAMP is nearly ineffective. Even for the loose conformational state the nucleotide derivatives exhibit considerable affinity. This allows DANSAMP to replace prebound ATP from UCP and relieve the inhibition of H+ transport by reversing the distribution of UCP from the tight into the loose conformational state. The pH dependence of the fraction of nucleotide binding in the tight state correlates closely with the pH dependence of the degree of H+ transport inhibition. Titration with DANS nucleotides of UCP incorporated into phospholipid vesicles revealed that over 70% of binding sites had an affinity comparable with that for the isolated UCP while the remaining sites displayed substantially lower affinity, due to nonhomogeneity of the reconstituted system. The sensitivity against trypsin digestion is inversely correlated with the fraction of nucleotide binding in the tight state. Whereas unsubstituted nucleotides and DANSGTP protect strongly against trypsinolysis, DANSATP and DANSADP do only partially, and DANSAMP does not at all. The counteracting influences of the DANS substitution are shown with DANSAMP, which has an affinity comparable to that of DANSATP or DANSADP but cannot form the tight inhibited complex. These data show that nucleotide binding only in the tight state is associated with a strong conformational change, which further causes an inhibition of H+ transport. In conclusion, UCP can exist in a loose noninhibited and a tight inhibited conformational state. The equilibrium between these two conformations is shifted to the tight state with unsubstituted nucleotides but remains to variable degrees in the loose state with DANS and DAN derivatives. The DANS group hinders progressively the transition to the tight state as the binding affinity of the underlying nucleotide decreases.

Nature of the masking of nucleotide-binding sites in brown adipose tissue mitochondria. Involvement of endogenous adenosine triphosphate.

Binding of the fluorescent nucleotide derivative 2'-O-dansyl GTP and purine nucleotides to brown adipose tissue mitochondria from hamster was studied. 2'-O-Dansyl GTP binds with enhanced fluorescence to the uncoupling protein (UCP) in the mitochondria, similar to the isolated protein. The fluorescence signal showed biphasic fast and slow increases. Treatment of the mitochondria with an anion exchanger (Dowex) increased the total fluorescence but decreased the slower phase. The biphasic fluorescence response was restored by incubation with only 1 microM ATP, indicating that residual bound ATP may be responsible for the observed slow phase. The binding of [14C]GTP and GDP also increased after Dowex treatment. The dissociation of bound [14C]ATP but not of bound [14C]ADP was slow and apparently limited the binding assays. Short incubation (5 min) resulted in a curvature of the Scatchard plot, where the 'high-affinity sites' correspond to the free UCP sites; GDP had apparently higher affinity than GTP. Dowex treatment and incubation for 60 min produced a more linear Scatchard plot. Under such conditions, one measures the maximal UCP-binding sites (1.2 mumol/g protein); GTP exhibited higher affinity (Kd = 0.64 microM) than GDP (Kd = 3.1 microM). Acute cold adaptation (40 min at 4 degrees C) of hamsters caused an increase by over 40% of [14C]GTP binding, as compared to the control warm-(28 degrees C)-adapted animals. Dowex treatment completely abolishes this unmasking/masking effect, where both mitochondria had identical binding capacity and affinity for GTP. The inhibition by purine nucleotides of H+ transport as measured by potassium-acetate-induced mitochondrial swelling was dependent on the incubation time. Diphosphates inhibited faster and triphosphates required longer incubation (10 min) but inhibited more strongly. A linear correlation between the mitochondrial swelling rate and GDP binding was observed for mitochondria with depleted endogenous ATP or with added ATP. These data indicate that residual bound ATP from the tissue is responsible for the masking phenomenon.

Fluorescent nucleotide derivatives as specific probes for the uncoupling protein: thermodynamics and kinetics of binding and the control by pH.

Fluorescent 2'-O-dansylated (DANS) purine nucleotides were synthesized. The fluorescence of the nucleotide derivatives is quenched in aqueous solutions but strongly enhanced on binding to the uncoupling protein (UCP) from brown adipose tissue mitochondria. The fluorescence enhancement was 30-, 10-, and 10-fold for DANSGTP, DANSATP, and DANSADP. One mole of DANS nucleotide binds to 1 mol of dimeric UCP. The binding affinity ranges from 10(5) to 10(8) M-1, similar to that of the unsubstituted nucleotides, while dansylation of AMP increases the affinity 50-fold. The pH dependence in the pKD/pH plots for the DANS nucleotides is basically similar to that for the unsubstituted nucleotides, i.e., for nucleoside diphosphates the slope delta pKD/delta pH < -1 at pH 5-6.5, = -1 at pH > 6.8, and only for triphosphates = -2 at pH > 7.2. Two different protonation sites with a pKH approximately 4 (Asp/Glu) and pKH approximately 7.2 (His), only for nucleoside triphosphates, are suggested to be involved in binding. The higher affinity of DANSGTP indicates additional participation in binding of the C-6 oxygen on the guanine. The binding as measured with the anion exchange method agrees with the fluorescence measurement for DANSGTP, whereas for the more loosely binding DANSATP it is 40% lower. This is interpreted in terms of tight/loose UCP-nucleotide complexes, 100% tight complex for DANSGTP (as well GTP or ATP) but 40% loose complex for DANSATP. By measuring the rapid kinetics using the fluorescence signal, the binding rate is found to be fast and fairly constant for the various nucleotides, whereas the dissociation is slow and strongly nucleotide dependent. The rates are pH dependent with delta pkon/delta pH = 1 for all the nucleotides and delta pkoff/delta pH = -1 for DANSNTP but more weakly with delta pkoff/delta pH < -0.5 for DANSADP and DAN-ATP. The pH dependence of the binding rate corresponds to a protonation at the carboxylate group (Glu/Asp). The high pH dependence of the dissociation rate only for DANSNTP is explained by deprotonation at the HisH+ which is involved only in nucleoside triphosphate binding. This is in line with the very strong pH dependence of nucleoside triphosphate affinity above pH 7 with a delta pKD/delta pH = -2 as an important regulatory mechanism for the H+ transport activity of UCP. The differences of the DANS nucleotides versus the DAN and unsubstituted nucleotides as well as the nucleoside tri- versus diphosphate are rationalized in a specific H+ dependent regulatory mechanism at the binding site.