Christophe Jardin

Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation.

The pUL97 protein kinase encoded by human cytomegalovirus is a multifunctional determinant of the efficiency of viral replication and phosphorylates viral as well as cellular substrate proteins. Here, we report that pUL97 is expressed in two isoforms with molecular masses of approximately 90 and 100 kDa. ORF UL97 comprises an unusual coding strategy in that five in-frame ATG start codons are contained within the N-terminal 157 aa. Site-directed mutagenesis, transient expression of point and deletion mutants and proteomic analyses accumulated evidence that the formation of the large and small isoforms result from alternative initiation of translation, with the start points being at amino acids 1 and 74, respectively. In vitro kinase assays demonstrated that catalytic activity, in terms of autophosphorylation and histone substrate phosphorylation, was indistinguishable for the two isoforms. An analysis of the intracellular distribution of pUL97 by confocal laser-scanning microscopy demonstrated that both isoforms have a pronounced nuclear localization. Surprisingly, mapping experiments performed to identify the nuclear localization signal (NLS) of pUL97 strongly suggest that the mechanism of nuclear transport is distinct for the two isoforms. While the extreme N terminus (large isoform) comprises a highly efficient, bipartite NLS (amino acids 6-35), a second sequence apparently conferring a less efficient mode of nuclear translocation was identified downstream of amino acid 74 (small and large isoforms). Taken together, the findings argue for a complex mechanism of nuclear translocation for pUL97 which might be linked with fine-regulatory differences between the two isoforms.

Channel Estimation for Diffusive Molecular Communications

In molecular communication (MC) systems, the expected number of molecules observed at the receiver over time after the instantaneous release of molecules by the transmitter is referred to as the channel impulse response (CIR). Knowledge of the CIR is needed for the design of detection and equalization schemes. In this paper, we present a training-based CIR estimation framework for MC systems, which aims at estimating the CIR based on the observed number of molecules at the receiver due to emission of a sequence of known numbers of molecules by the transmitter. Thereby, we distinguish two scenarios depending on whether or not statistical channel knowledge is available. In particular, we derive maximum likelihood and least sum of square errors estimators, which do not require any knowledge of the channel statistics. For the case, when statistical channel knowledge is available, the corresponding maximum a posteriori and linear minimum mean square error estimators are provided. As performance bound, we derive the classical Cramer Rao (CR) lower bound, valid for any unbiased estimator, which does not exploit statistical channel knowledge, and the Bayesian CR lower bound, valid for any unbiased estimator, which exploits statistical channel knowledge. Finally, we propose the optimal and suboptimal training sequence designs for the considered MC system. Simulation results confirm the analysis and compare the performance of the proposed estimation techniques with the respective CR lower bounds.

DNA binding by Corynebacterium glutamicum TetR-type transcription regulator AmtR

BackgroundThe TetR family member AmtR is the central regulator of nitrogen starvation response in Corynebacterium glutamicum. While the AmtR regulon was physiologically characterized in great detail up to now, mechanistic questions of AmtR binding were not addressed. This study presents a characterization of functionally important amino acids in the DNA binding domain of AmtR and of crucial nucleotides in the AmtR recognition motif.ResultsSite-directed mutagenesis, the characterization of corresponding mutant proteins by gel retardation assays and surface plasmon resonance and molecular modelling revealed several amino acids, which are directly involved in DNA binding, while others have more structural function. Furthermore, we could show that the spacing of the binding motif half sites is crucial for repression of transcription by AmtR.ConclusionAlthough the DNA binding domain of TetR-type repressors is highly conserved and a core binding motif was identified for AmtR and TetR(D), the AmtR binding domain shows individual properties compared to other TetR proteins. Besides by distinct amino acids of AmtR, DNA binding is influenced by nucleotides not only of the conserved binding motif but also by spacing nucleotides in C. glutamicum.

Nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding

The multifunctional protein kinase pUL97 of human cytomegalovirus (HCMV) strongly determines the efficiency of virus replication. Previously, the existence of two pUL97 isoforms that arise from alternative translational initiation and show a predominant nuclear localization was described. Two bipartite nuclear localization sequences, NLS1 and NLS2, were identified in the N terminus of the large isoform, whilst the small isoform exclusively contained NLS2. The current study found the following: (i) pUL97 nuclear localization in HCMV-infected primary fibroblasts showed accumulations in virus replication centres and other nuclear sections; (ii) in a quantitative evaluation system for NLS activity, the large isoform showed higher efficiency of nuclear translocation than the small isoform; (iii) NLS1 was mapped to aa 6-35 and NLS2 to aa 190-213; (iv) using surface plasmon resonance spectroscopy, the binding of both NLS1 and NLS2 to human importin-α was demonstrated, stressing the importance of individual arginine residues in the bipartite consensus motifs; (v) nuclear magnetic resonance spectroscopy of pUL97 peptides confirmed an earlier statement about the functional requirement of NLS1 embedding into an intact α-helical structure; and (vi) a bioinformatics investigation of the solvent-accessible surface suggested a high accessibility of NLS1 and an isoform-specific, variable accessibility of NLS2 for interaction with importin-α. Thus, the nucleocytoplasmic transport mechanism of the isoforms appeared to be differentially regulated, and this may have consequences for isoform-dependent functions of pUL97 during virus replication.

Insight into the phosphoryl transfer of the Escherichia coli glucose phosphotransferase system from QM/MM simulations.

Phosphoryl transfer is a key reaction in many aspects of metabolism, gene regulation, and signal transduction. One prominent example is the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which represents an integral part of the bacterial sugar metabolism. The transfer between the enzymes IIA (Glc) and IIB (Glc) in the glucose-specific branch of the PTS is of particular interest due to the unusual combination of donor and acceptor residues involved in phosphoryl transfer: The phosphoryl group is initially attached to the Nepsilon2 atom of His 90 in IIA (Glc) and then transferred to the S gamma atom of Cys 35 in IIB (Glc). To gain insight into the details of the transfer mechanism, we have performed a QM/MM simulation which treats the entire active site quantum-mechanically. The transfer has a high dissociative character, and the Nepsilon2-P bond gets immediately destabilized after complex formation by numerous interactions formed between residues of IIB (Glc) and the phosphoryl group. The final formation of a tight S gamma-P bond is accompanied by a reorientation of the side chain of the phosphoryl donor. This reorientation results in the loss of interaction between the imidazole ring and the phosphate group thus hindering the reverse transfer. A comparison to the transfer in protein tyrosine phosphatases, which also use a cysteine as acceptor of the phosphoryl group, reveals significant similarities in the conformation of the active site, the energy profile of the reaction, and in the pattern of interactions that stabilize the phosphoryl group during the transfer.

A Molecular Model for the Differential Activation of STAT3 and STAT6 by the Herpesviral Oncoprotein Tip

Constitutive STAT signaling provides growth promoting signals in many forms of malignancy. We performed molecular modeling and molecular dynamics studies of the interaction between the regulatory Src homology 2 (SH2) domains of STAT3 and 6 with phosphorylated peptides of the herpesviral oncoprotein Tip, which facilitates Src kinase mediated STAT-activation and T cell proliferation. The studies give insight into the ligand binding specificity of the STAT SH2 domains and provide the first model for the differential activation of STAT3 or STAT6 by two distinct regions of the viral Tip protein. The biological relevance of the modeled interactions was then confirmed by activation studies using corresponding recombinant oncoproteins, and finally by respective recombinant viruses. The functional data give experimental validation of the molecular dynamics study, and provide evidence for the involvement of STAT6 in the herpesvirus induced T cell proliferation.

Binding properties of SUMO-interacting motifs (SIMs) in yeast

Small ubiquitin-like modifier (SUMO) conjugation and interaction play an essential role in many cellular processes. A large number of yeast proteins is known to interact non-covalently with SUMO via short SUMO-interacting motifs (SIMs), but the structural details of this interaction are yet poorly characterized. In the present work, sequence analysis of a large dataset of 148 yeast SIMs revealed the existence of a hydrophobic core binding motif and a preference for acidic residues either within or adjacent to the core motif. Thus the sequence properties of yeast SIMs are highly similar to those described for human. Molecular dynamics simulations were performed to investigate the binding preferences for four representative SIM peptides differing in the number and distribution of acidic residues. Furthermore, the relative stability of two previously observed alternative binding orientations (parallel, antiparallel) was assessed. For all SIMs investigated, the antiparallel binding mode remained stable in the simulations and the SIMs were tightly bound via their hydrophobic core residues supplemented by polar interactions of the acidic residues. In contrary, the stability of the parallel binding mode is more dependent on the sequence features of the SIM motif like the number and position of acidic residues or the presence of additional adjacent interaction motifs. This information should be helpful to enhance the prediction of SIMs and their binding properties in different organisms to facilitate the reconstruction of the SUMO interactome.

Channel estimation techniques for diffusion-based molecular communications

In molecular communication (MC) systems, the expected number of molecules observed at the receiver over time after the instantaneous release of molecules by the transmitter is referred to as the channel impulse response (CIR). Knowledge of the CIR is needed for the design of detection and equalization schemes. In this paper, we present a training-based CIR estimation framework for MC systems which aims at estimating the CIR based on the observed number of molecules at the receiver due to emission of a sequence of known numbers of molecules by the transmitter. In particular, we derive maximum likelihood (ML) and least sum of square errors (LSSE) estimators. We also study the Cramer Rao (CR) lower bound and training sequence design for the considered system. Simulation results confirm the analysis and compare the performance of the proposed estimation techniques with the CR lower bound.

Identification of the Structural Features that Mediate Binding Specificity in the Recognition of STAT Proteins by Dual-Specificity Phosphatases

Abstract Inactivation of signal transducers and activators of transcription (STAT) proteins is regulated by dual-specificity phosphatases (DSPs) with high substrate specificity. Although experiments have provided useful information about the phosphatase activity and the specificity for STATs, there is up-to-date no data at a molecular level to explain the specific recognition of STAT substrates by this subfamily of phosphatases. Here, a combined approach of molecular modeling, docking and molecular dynamics simulations was used to address the binding between DSPs and their STAT substrates. We identified a binding interface at the protein tyrosine phosphatase (PTP) domain of the DSP VHR that interacts with the SH2-domain of STAT5. This finding is consistent with previous mutational data and supports a “two-step” mechanism for the dephosphorylation event. Application of the same approach suggests the presence of a similar interface between the viral DSP VH1 and STAT1. Furthermore, the interaction network at this interface provides an explanation for the specificity of the DSP-STAT recognition.

Confidence intervals for the mutual information

By combining a bound on the absolute value of the difference of mutual information between two joint probablity distributions with a fixed variational distance, and a bound on the probability of a maximal deviation in variational distance between a true joint probability distribution and an empirical joint probability distribution, confidence intervals for the mutual information of two random variables with finite alphabets are established. Different from previous results, these intervals do not need any assumptions on the distribution and the sample size.