Role of receptor-attached phosphates in binding of visual and non-visual arrestins to G protein-coupled receptors

Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in β-strand I was shown to disrupt the interaction of α-helix I, β-strand I, and the C-tail of visual arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins in which the K2A mutations had little effect on net BRET(max) values for the M2 muscarinic acetylcholine (M2R), β(2)-adrenergic (β(2)AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual arrestins normally, whereas phosphorylation-deficient β(2)AR mutants bound arrestins at 20-50% of the level of wild type β(2)AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. Although arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of arrestin-2 and -3 is much greater in the case of β(2)AR than M2R and D2 dopamine receptor.     

Loss and Recovery of Genetic Diversity in Adapting Populations of HIV

The evolution of drug resistance in HIV occurs by the fixation of specific, well-known, drug-resistance mutations, but the underlying population genetic processes are not well understood. By analyzing within-patient longitudinal sequence data, we make four observations that shed a light on the underlying processes and allow us to infer the short-term effective population size of the viral population in a patient. Our first observation is that the evolution of drug resistance usually occurs by the fixation of one drug-resistance mutation at a time, as opposed to several changes simultaneously. Second, we find that these fixation events are accompanied by a reduction in genetic diversity in the region surrounding the fixed drug-resistance mutation, due to the hitchhiking effect. Third, we observe that the fixation of drug-resistance mutations involves both hard and soft selective sweeps. In a hard sweep, a resistance mutation arises in a single viral particle and drives all linked mutations with it when it spreads in the viral population, which dramatically reduces genetic diversity. On the other hand, in a soft sweep, a resistance mutation occurs multiple times on different genetic backgrounds, and the reduction of diversity is weak. Using the frequency of occurrence of hard and soft sweeps we estimate the effective population size of HIV to be ( confidence interval ). This number is much lower than the actual number of infected cells, but much larger than previous population size estimates based on synonymous diversity. We propose several explanations for the observed discrepancies. Finally, our fourth observation is that genetic diversity at non-synonymous sites recovers to its pre-fixation value within 18 months, whereas diversity at synonymous sites remains depressed after this time period. These results improve our understanding of HIV evolution and have potential implications for treatment strategies.     

Microbial Growth and Carbon Use Efficiency in the Rhizosphere and Root-Free Soil

Plant-microbial interactions alter C and N balance in the rhizosphere and affect the microbial carbon use efficiency (CUE)–the fundamental characteristic of microbial metabolism. Estimation of CUE in microbial hotspots with high dynamics of activity and changes of microbial physiological state from dormancy to activity is a challenge in soil microbiology. We analyzed respiratory activity, microbial DNA content and CUE by manipulation the C and nutrients availability in the soil under Beta vulgaris. All measurements were done in root-free and rhizosphere soil under steady-state conditions and during microbial growth induced by addition of glucose. Microorganisms in the rhizosphere and root-free soil differed in their CUE dynamics due to varying time delays between respiration burst and DNA increase. Constant CUE in an exponentially-growing microbial community in rhizosphere demonstrated the balanced growth. In contrast, the CUE in the root-free soil increased more than three times at the end of exponential growth and was 1.5 times higher than in the rhizosphere. Plants alter the dynamics of microbial CUE by balancing the catabolic and anabolic processes, which were decoupled in the root-free soil. The effects of N and C availability on CUE in rhizosphere and root-free soil are discussed.     

Structural and functional characterization of an anti‐West Nile virus monoclonal antibody and its single‐chain variant produced in glycoengineered plants

Previously, our group engineered a plant‐derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single‐chain variable fragment (scFv) fused to the heavy chain constant domains (C_H) of human IgG (pE16scFv‐C_H). pE16 and pE16scFv‐C_H were expressed and assembled efficiently in Nicotiana benthamiana ?XF plants, a glycosylation mutant lacking plant‐specific N‐glycan residues. Glycan analysis revealed that ?XF plant‐derived pE16scFv‐C_H (?XFpE16scFv‐C_H) and pE16 (?XFpE16) both displayed a mammalian glycosylation profile. ?XFpE16 and ?XFpE16scFv‐C_H demonstrated equivalent antigen‐binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell‐produced E16 (mE16). A single dose of ?XFpE16 or ?XFpE16scFv‐C_H protected mice against WNV‐induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single‐chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti‐WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N‐glycosylation. This platform may lead to a more robust and cost‐effective production of antibody‐based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases.     

Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

The non-visual arrestins, arrestin-2 and arrestin-3, belong to a small family of multifunctional cytosolic proteins. Non-visual arrestins interact with hundreds of G protein-coupled receptors (GPCRs) and regulate GPCR desensitization by binding active phosphorylated GPCRs and uncoupling them from heterotrimeric G proteins. Recently, non-visual arrestins have been shown to mediate G protein-independent signaling by serving as adaptors and scaffolds that assemble multiprotein complexes. By recruiting various partners, including trafficking and signaling proteins, directly to GPCRs, non-visual arrestins connect activated receptors to diverse signaling pathways. To investigate arrestin-mediated signaling, a structural understanding of arrestin activation and interaction with GPCRs is essential. Here we identified global and local conformational changes in the non-visual arrestins upon binding to the model GPCR rhodopsin. To detect conformational changes, pairs of spin labels were introduced into arrestin-2 and arrestin-3, and the interspin distances in the absence and presence of the receptor were measured by double electron electron resonance spectroscopy. Our data indicate that both non-visual arrestins undergo several conformational changes similar to arrestin-1, including the finger loop moving toward the predicted location of the receptor in the complex as well as the C-tail release upon receptor binding. The arrestin-2 results also suggest that there is no clam shell-like closure of the N- and C-domains and that the loop containing residue 136 (homolog of 139 in arrestin-1) has high flexibility in both free and receptor-bound states.     

Applications of dual-color fluorescence cross-correlation spectroscopy in antibody binding studies

Two-photon dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) was applied to study the binding interactions of monoclonal antibodies (mAbs) and protein antigens. We measured the binding constant of the interaction of a 32-amino acid brain natriuretic peptide (BNP) with a mAbs and demonstrated the utility of DC-FCCS in studies of antibody sandwiches, trimolecular formations, where two different antibodies bind the same antigen simultaneously. We also show the use of DC-FCCS for monitoring competitive displacement of the labeled antibody in antibody-antigen complexes and subsequent determination of the pertinent dissociation rate (off-rate). The off-rate measurements were performed for two mAbs toward tissue inhibitor 1 of metalloproteinases (TIMP-1). From a methodological perspective, selection of the best labeling protocols and careful optimization of the FCCS instrumentation are essential to achieve the highest cross-correlation signal. When working in vitro, it is practical to generate a complete binding curve using the normalized cross-correlation signal and then fit the experimental points to a binding model. DC-FCCS offers the sensitivity and all other advantages of a solution phase fluorescence-based technique. For systems containing proteins of a similar size that interact without substantial changes in the fluorescence intensity, DC-FCCS serves as a preferred means of measuring solution phase binding constants.     

Study of 1-deoxy-1-(indol-3-yl)-L-sorbose, 1-deoxy-1-(indol-3-yl)-L-tagatose,and their analogs

Alkaline degradation of the ascorbigen 2-C-[(indol-3-yl)methyl]-alpha-L-xylo-hex-3-ulofuranosono-1,4-lactone (1a) led to a mixture of 1-deoxy-1-(indol-3-yl)-L-sorbose (2a) and 1-deoxy-1-(indol-3-yl)-L-tagatose (3a). The mixture of diastereomeric ketoses underwent acetylation and pyranose ring opening under the action of acetic anhydride in pyridine in the presence of 4-dimethylaminopyridine (DMAP) with the formation of a mixture of (E)-2,3,4,5,6-penta-O-acetyl-1-deoxy-1-(indol-3-yl)-L-xylo-hex-1-enitol (4a) and (E)-2,3,4,5,6-penta-O-acetyl-1-deoxy-1-(indol-3-yl)-L-lyxo-hex-1-enitol (5a), which were separated chromatographically. Deacetylation of 4a or 5a afforded cyclised tetrols, tosylation of which in admixture resulted in 1-deoxy-1-(indol-3-yl)-3,5-di-O-tosyl-alpha-L-sorbopyranose (12a) and 1-deoxy-1-(indol-3-yl)-4,5-di-O-tosyl-alpha-L-tagatopyranose (13a). Under alkaline conditions 13a readily formed 2-hydroxy-4-hydroxymethyl-3-(indol-3-yl)cyclopenten-2-one (15a) in 90% yield. Similar transformations were performed for N-methyl- and N-methoxyindole derivatives.     

Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cellular redox activity of coenzyme Q10: effect of CoQ10 supplementation on human skeletal muscle

In this paper, we report results obtained from a continuing clinical trial on the effect of coenzyme Q 10 (CoQ 10 ) administration on human vastus lateralis (quadriceps) skeletal muscle. Muscle samples, obtained from aged individuals receiving placebo or CoQ 10 supplementation (300 mg per day for four weeks prior to hip replacement surgery) were analysed for changes in gene and protein expression and in muscle fibre type composition. Microarray analysis (Affymetrix U95A human oligonucleotide array) using a change in gene expression of 1.8-fold or greater as a cutoff point, demonstrated that a total of 115 genes were differentially expressed in six subject comparisons. In the CoQ 10 -treated subjects, 47 genes were up-regulated and 68 down-regulated in comparison with placebo-treated subjects. Restriction fragment differential display analysis showed that over 600 fragments were differentially expressed using a 2.0-fold or greater change in expression as a cutoff point. Proteome analysis revealed that, of the high abundance muscle proteins detected (2086 ±115), the expression of 174 proteins was induced by CoQ 10 while 77 proteins were repressed by CoQ 10 supplementation. Muscle fibre types were also affected by CoQ 10 treatment; CoQ 10 -treated individuals showed a lower proportion of type I (slow twitch) fibres and a higher proportion of type IIb (fast twitch) fibres, compared to age-matched placebo-treated subjects. The data suggests that CoQ 10 treatment can act to influence the fibre type composition towards the fibre type profile generally found in younger individuals. Our results led us to the conclusion that coenzyme Q 10 is a gene regulator and consequently has wide-ranging effects on over-all tissue metabolism. We develop a comprehensive hypothesis that CoQ 10 plays a major role in the determination of membrane potential of many, if not all, sub-cellular membrane systems and that H 2 O 2 arising from the activities of CoQ 10 acts as a second messenger for the modulation of gene expression and cellular metabolism.