In Vivo Binding and Retention of CD4-Specific DARPin 57.2 in Macaques

The recently described Designed Ankyrin Repeat Protein (DARPin) technology can produce highly selective ligands to a variety of biological targets at a low production cost.To investigate the in vivo use of DARPins for future application to novel anti-HIV strategies, we identified potent CD4-specific DARPins that recognize rhesus CD4 and followed the fate of intravenously injected CD4-specific DARPin 57.2 in rhesus macaques. The human CD4-specific DARPin 57.2 bound macaque CD4(+) cells and exhibited potent inhibitory activity against SIV infection in vitro. DARPin 57.2 or the control E3_5 DARPin was injected into rhesus macaques and the fate of cell-free and cell-bound CD4-specific DARPin was evaluated. DARPin-bound CD4(+) cells were detected in the peripheral blood as early as 30 minutes after the injection, decreasing within 6 hours and being almost undetectable within 24 hours. The amount of DARPin bound was dependent on the amount of DARPin injected. CD4-specific DARPin was also detected on CD4(+) cells in the lymph nodes within 30 minutes, which persisted with similar kinetics to blood. More extensive analysis using blood revealed that DARPin 57.2 bound to all CD4(+) cell types (T cells, monocytes, dendritic cells) in vivo and in vitro with the amount of binding directly proportional to the amount of CD4 on the cell surface. Cell-free DARPins were also detected in the plasma, but were rapidly cleared from circulation.We demonstrated that the CD4-specific DARPin can rapidly and selectively bind its target cells in vivo, warranting further studies on possible clinical use of the DARPin technology

Integrative inference of gene-regulatory networks in Escherichia coli using information theoretic concepts and sequence analysis

<p>Abstract</p> <p>Background</p> <p>Although <it>Escherichia coli </it>is one of the best studied model organisms, a comprehensive understanding of its gene regulation is not yet achieved. There exist many approaches to reconstruct regulatory interaction networks from gene expression experiments. Mutual information based approaches are most useful for large-scale network inference.</p> <p>Results</p> <p>We used a three-step approach in which we combined gene regulatory network inference based on directed information (DTI) and sequence analysis. DTI values were calculated on a set of gene expression profiles from 19 time course experiments extracted from the Many Microbes Microarray Database. Focusing on influences between pairs of genes in which one partner encodes a transcription factor (TF) we derived a network which contains 878 TF - gene interactions of which 166 are known according to RegulonDB. Afterward, we selected a subset of 109 interactions that could be confirmed by the presence of a phylogenetically conserved binding site of the respective regulator. By this second step, the fraction of known interactions increased from 19% to 60%. In the last step, we checked the 44 of the 109 interactions not yet included in RegulonDB for functional relationships between the regulator and the target and, thus, obtained ten TF - target gene interactions. Five of them concern the regulator LexA and have already been reported in the literature. The remaining five influences describe regulations by Fis (with two novel targets), PhdR, PhoP, and KdgR. For the validation of our approach, one of them, the regulation of lipoate synthase (LipA) by the pyruvate-sensing pyruvate dehydrogenate repressor (PdhR), was experimentally checked and confirmed.</p> <p>Conclusions</p> <p>We predicted a set of five novel TF - target gene interactions in <it>E. coli</it>. One of them, the regulation of <it>lipA </it>by the transcriptional regulator PdhR was validated experimentally. Furthermore, we developed DTInfer, a new R-package for the inference of gene-regulatory networks from microarrays using directed information.</p

ANKRD54 preferentially selects Bruton's Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library

Bruton's Tyrosine Kinase (BTK) is a cytoplasmic protein tyrosine kinase with a fundamental role in B-lymphocyte development and activation. The nucleocytoplasmic shuttling of BTK is specifically modulated by the Ankyrin Repeat Domain 54 (ANKRD54) protein and the interaction is known to be exclusively SH3-dependent. To identify the spectrum of the ANKRD54 SH3-interactome, we applied phage-display screening of a library containing all the 296 human SH3 domains. The BTK-SH3 domain was the prime interactor. Quantitative western blotting analysis demonstrated the accuracy of the screening procedure. Revealing the spectrum and specificity of ANKRD54-interactome is a critical step toward functional analysis in cells and tissues.Peer reviewe

A Novel RNA-Recognition-Motif Protein Is Required for Premeiotic G1/S-Phase Transition in Rice (Oryza sativa L.)

The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1), though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species

The effect of prior walking on coronary heart disease risk markers in South Asian and European men.

Purpose: Heart disease risk is elevated in South Asians possibly due to impaired postprandial metabolism. Running has been shown to induce greater reductions in postprandial lipaemia in South Asian than European men but the effect of walking in South Asians is unknown. Methods: Fifteen South Asian and 14 White European men aged 19-30 years completed two, 2-d trials in a randomised crossover design. On day 1, participants rested (control) or walked for 60 min at approximately 50% maximum oxygen uptake (exercise). On day 2, participants rested and consumed two high fat meals over a 9h period during which 14 venous blood samples were collected. Results: South Asians exhibited higher postprandial triacylglycerol (geometric mean (95% confidence interval) 2.29(1.82 to 2.89) vs. 1.54(1.21 to 1.96) mmol·L-1·hr-1), glucose (5.49(5.21 to 5.79) vs. 5.05(4.78 to 5.33) mmol·L-1·hr-1), insulin (32.9(25.7 to 42.1) vs. 18.3(14.2 to 23.7) µU·mL-1·hr-1) and interleukin-6 (2.44(1.61 to 3.67) vs. 1.04(0.68 to 1.59) pg·mL-1·hr-1) than Europeans (all ES ≥ 0.72, P≤0.03). Between-group differences in triacylglycerol, glucose and insulin were not significant after controlling for age and percentage body fat. Walking reduced postprandial triacylglycerol (1.79(1.52 to 2.12) vs. 1.97(1.67 to 2.33) mmol·L-1·hr-1) and insulin (21.0(17.0 to 26.0) vs. 28.7(23.2 to 35.4) µU·mL-1·hr-1) (all ES ≥ 0.23. P≤0.01), but group differences were not significant. Conclusions: Healthy South Asians exhibited impaired postprandial metabolism compared with White Europeans, but these differences were diminished after controlling for potential confounders. The small-moderate reduction in postprandial triacylglycerol and insulin after brisk walking was not different between the ethnicities

Structure-Function Relationship of Cytoplasmic and Nuclear IκB Proteins: An In Silico Analysis

Cytoplasmic IκB proteins are primary regulators that interact with NF-κB subunits in the cytoplasm of unstimulated cells. Upon stimulation, these IκB proteins are rapidly degraded, thus allowing NF-κB to translocate into the nucleus and activate the transcription of genes encoding various immune mediators. Subsequent to translocation, nuclear IκB proteins play an important role in the regulation of NF-κB transcriptional activity by acting either as activators or inhibitors. To date, molecular basis for the binding of IκBα, IκBβ and IκBζ along with their partners is known; however, the activation and inhibition mechanism of the remaining IκB (IκBNS, IκBε and Bcl-3) proteins remains elusive. Moreover, even though IκB proteins are structurally similar, it is difficult to determine the exact specificities of IκB proteins towards their respective binding partners. The three-dimensional structures of IκBNS, IκBζ and IκBε were modeled. Subsequently, we used an explicit solvent method to perform detailed molecular dynamic simulations of these proteins along with their known crystal structures (IκBα, IκBβ and Bcl-3) in order to investigate the flexibility of the ankyrin repeat domains (ARDs). Furthermore, the refined models of IκBNS, IκBε and Bcl-3 were used for multiple protein-protein docking studies for the identification of IκBNS-p50/p50, IκBε-p50/p65 and Bcl-3-p50/p50 complexes in order to study the structural basis of their activation and inhibition. The docking experiments revealed that IκBε masked the nuclear localization signal (NLS) of the p50/p65 subunits, thereby preventing its translocation into the nucleus. For the Bcl-3- and IκBNS-p50/p50 complexes, the results show that Bcl-3 mediated transcription through its transactivation domain (TAD) while IκBNS inhibited transcription due to its lack of a TAD, which is consistent with biochemical studies. Additionally, the numbers of identified flexible residues were equal in number among all IκB proteins, although they were not conserved. This could be the primary reason for their binding partner specificities

H3K9me-Independent Gene Silencing in Fission Yeast Heterochromatin by Clr5 and Histone Deacetylases

Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress

The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated, and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales

Novel Structural Components of the Ventral Disc and Lateral Crest in Giardia intestinalis

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment

Anchoring skeletal muscle development and disease: the role of ankyrin repeat domain containing proteins in muscle physiology

The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies