Molecular Simulation Approaches to Proteins Structure and Dynamics and to Ligand Design

Molecular simulations approaches are powerful tools for structural biology and drug discovery. They provide additional and complementary information on structure, dynamics and energetics of biomolecules whose structures have been determined experimentally [1,2,3,4,5,6,7]. In particular, Molecular Dynamics (MD) simulations [8], along with elastic network analysis [9], offer insights into molecular fluctuations, conformational changes and allosteric mechanisms. In addition, molecular simulation can be used to design novel and potent ligands to a specific target (either a protein or DNA) as well as to estimate ligands potency [10,11]. Attempts at predicting protein structures using bioinformatics and MD are also increasingly successful [12,13,14,15], as well as approaches that use solely simulation tools [16]. The development of new algorithms and the continuously growing computer power currently allow for the simulation of more and more complex biological systems, such as protein aggregates [7,17,18,19,20] and protein/DNA complexes [21]. In this context, a number of theoretical techniques (namely molecular dynamics simulations, elastic network analysis, electrostatic modeling and binding energy predictions) have here been applied to the study of specific proteins. On the basis of X-ray protein structures, molecular simulations have provided a detailed description of internal motions and interactions, which are not evident from the experimental data and have functional implications. First, we have used MD to investigate structural features, focusing on the differences between the solid state and the aqueous solution structures. Over 80% of data in the PDB [22] are X-ray structures, making protein crystallography the major resource in structural biology. Nevertheless, in a few cases, the structural details might be affected by environmental features, such as the presence of small compounds in the buffering solution and/or crystal packing contacts due to the periodic lattice. Here, a comparative MD study has been performed on the Catabolite Activator Protein (CAP), in both the crystal phase and in the aqueous solution. CAP is a bacterial DNA-binding transcription regulator whose activity is controlled by the binding of the intracellular mediator cyclic Adenosine MonoPhosphate (cAMP). CAP is a homodimeric protein and each subunit is formed by a cyclic nucleotide- and a DNA-binding domain. Inspection of the available CAP X-ray structure within the crystal environment [23] suggests that packing contacts do affect the native conformation of the ligand activated protein. Anticipating our results, we have found that indeed the conformation of the protein in solution is different, and that these differences may play a role for CAP biological function. Next, we have used molecular simulations to target structural flexibility. Conformational fluctuations often play a key role for the protein function and MD simulations can provide information on large-scale concerted motions of proteins [24,25,26]. We have addressed this point in the context of the Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) cation channel. The tetrameric HCN channels are opened by membrane hyperpolarization, while their activation is allosterically modulated by the binding of cAMP in the cytoplasm. The cytoplasmic part of the HCN2 channel, which is responsible for the channel modulation, has been here investigated by MD simulations and elastic network analysis, on the basis of the available X-ray structure [27], to earn new insights into the molecular mechanism triggered by cAMP. We have found that, in the presence of cAMP, the protein undergoes a quaternary structure oscillation, in which each subunit moves as a rigid body. This fluctuation, which is not observed in the absence of cAMP, could facilitate the channel opening transition. Finally, we have moved our attention to an issue relevant for structure-based drug design. Within a long-standing collaboration with Prof. Cattaneo\u2019s lab (SISSA and Motivations and Summary 7 Layline Genomics), our group has been interested in the design of mimics of proteins involved in the biochemical pathways that lead to the Alzheimer\u2019s disease. Here, on the basis of structural information [28], we have designed a peptide that could specifically target trkA, the high affinity receptor of the Nerve Growth Factor (NGF), which is a protein that plays a critical role for the development, survival and maintenance of neurons in the vertebrate nervous system and activates signaling pathways related to neuroprotection. The results of this research will be tested at the Prof. Cattaneo\u2019s Lab in order to validate the theoretical findings and assess the potency and the effects of such a ligand

Amino acid empirical contact energy definitions for fold recognition in the space of contact maps

BACKGROUND: Contradicting evidence has been presented in the literature concerning the effectiveness of empirical contact energies for fold recognition. Empirical contact energies are calculated on the basis of information available from selected protein structures, with respect to a defined reference state, according to the quasi-chemical approximation. Protein-solvent interactions are estimated from residue solvent accessibility. RESULTS: In the approach presented here, contact energies are derived from the potential of mean force theory, several definitions of contact are examined and their performance in fold recognition is evaluated on sets of decoy structures. The best definition of contact is tested, on a more realistic scenario, on all predictions including sidechains accepted in the CASP4 experiment. In 30 out of 35 cases the native structure is correctly recognized and best predictions are usually found among the 10 lowest energy predictions. CONCLUSION: The definition of contact based on van der Waals radii of alpha carbon and side chain heavy atoms is seen to perform better than other definitions involving only alpha carbons, only beta carbons, all heavy atoms or only backbone atoms. An important prerequisite for the applicability of the approach is that the protein structure under study should not exhibit anomalous solvent accessibility, compared to soluble proteins whose structure is deposited in the Protein Data Bank. The combined evaluation of a solvent accessibility parameter and contact energy allows for an effective gross screening of predictive models

Single base mismatches in the mRNA target site allow specific seed region-mediated off-target binding of siRNA targeting human coagulation factor 7

We have analyzed the off-target activity of two siRNAs (F7-1, F7-2) that knock-down human blood coagulation factor 7 mRNA. F7-1 modulates a significant number of non-target transcripts while F7-2 shows high selectivity for the target transcript under various experimental conditions. The 3′-UTRs of all F7-1 off-target genes show statistically significant enrichment of the reverse complement of the F7-1 siRNA seed region located in the guide strand. Seed region enrichment was confirmed in off-target transcripts modulated by siRNA targeting the glucocorticoid receptor. To investigate how these sites contribute to off-target recognition of F7-1, we employed CXCL5 transcript as model system because it contains five F7-1 seed sequence motifs with single base mismatches. We show by transient transfection of reporter gene constructs into HEK293 cells that three out of five sites located in the 3′-UTR region are required for F7-1 off-target activity. For further mechanistic dissection, the sequences of these sites were synthesized and inserted either individually or joined in dimeric or trimeric constructs. Only the fusion constructs were silenced by F7-1 while the individual sites had no off-target activity. Based on F7-1 as a model, a single mismatch between the siRNA seed region and mRNA target sites is tolerated for target recognition and the CXCL5 data suggest a requirement for binding to multiple target sites in off-target transcripts

High throughput transcriptome analysis of lipid metabolism in Syrian hamster liver in absence of an annotated genome

Background: Whole transcriptome analyses are an essential tool for understanding disease mechanisms. Approaches based on next-generation sequencing provide fast and affordable data but rely on the availability of annotated genomes. However, there are many areas in biomedical research that require non-standard animal models for which genome information is not available. This includes the Syrian hamster Mesocricetus auratus as an important model for dyslipidaemia because it mirrors many aspects of human disease and pharmacological responses. We show that complementary use of two independent next generation sequencing technologies combined with mapping to multiple genome databases allows unambiguous transcript annotation and quantitative transcript imaging. We refer to this approach as ``triple match sequencing{''} (TMS). Results: Contigs assembled from a normalized Roche 454 hamster liver library comprising 1.2 million long reads were used to identify 10'800 unique transcripts based on homology to RefSeq database entries from human, mouse, and rat. For mRNA quantification we mapped 82 million SAGE tags (SOLiD) from the same RNA source to the annotated hamster liver transcriptome contigs. We compared the liver transcriptome of hamster with equivalent data from human, rat, minipig, and cynomolgus monkeys to highlight differential gene expression with focus on lipid metabolism. We identify a cluster of five genes functionally related to HDL metabolism that is expressed in human, cynomolgus, minipig, and hamster but lacking in rat as a non-responder species for lipid lowering drugs. Conclusions: The TMS approach is suited for fast and inexpensive transcript profiling in cells or tissues of species where a fully annotated genome is not available. The continuously growing number of well annotated reference genomes will further empower reliable transcript identification and thereby raise the utility of the method for any species of interest

Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog

<p><b>Background</b></p> <p>A novel fluorescent cAMP analog (8-[Pharos-575]- adenosine-3', 5'-cyclic monophosphate) was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Iα, PKA-IIα, PKA-IIβ) in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging.</p> <p><b>Results</b></p> <p>The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%. Incubation of the compound to RIIα and RIIβ subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIα. In vitro binding of the compound to RIα subunit and activation of the PKA-Iα holoenzyme was essentially equivalent to cAMP; RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the holoenzymes compared to cAMP. The cellular uptake of the fluorescent analog was investigated by cAMP indicators. It was estimated that about 7 μM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 μM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIα sensor.</p> <p><b>Conclusion</b></p> <p>The novel analog combines good membrane permeability- comparable to 8-Br-cAMP – with superior spectral properties of a modern, red-shifted fluorophore. GFP-tagged regulatory subunits of PKA and the analog co-localized. Furthermore, it is a potent, PDE-resistant activator of PKA-I and -II, suitable for in vitro applications and spatial distribution evaluations in living cells.</p&gt

Functional analysis and transcriptional output of the Göttingen minipig genome

In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.; Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies.; Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed

Sodium Iodate-Induced Degeneration Results in Local Complement Changes and Inflammatory Processes in Murine Retina

Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1β, il-33 and tgf-β were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy

Conversion systems for braking energy recovery in 3 kVDc railway lines

This paper deals with the energy saving given by the recovery of the braking energy coming from the trains in railway transportation systems. In particular, the paper is focused on the discussion about the conversion systems that are needed to achieve the power flow control from the braking trains to the station load through the energy exchange with the storage devices. These converters are actually realized to form a full-scale prototype applied to a regional 3 kVDC railway line