Patterns of evolution of host proteins involved in retroviral pathogenesis.

BACKGROUND: Evolutionary analysis may serve as a useful approach to identify and characterize host defense and viral proteins involved in genetic conflicts. We analyzed patterns of coding sequence evolution of genes with known (TRIM5alpha and APOBEC3G) or suspected (TRIM19/PML) roles in virus restriction, or in viral pathogenesis (PPIA, encoding Cyclophilin A), in the same set of human and non-human primate species. RESULTS AND CONCLUSION: This analysis revealed previously unidentified clusters of positively selected sites in APOBEC3G and TRIM5alpha that may delineate new virus-interaction domains. In contrast, our evolutionary analyses suggest that PPIA is not under diversifying selection in primates, consistent with the interaction of Cyclophilin A being limited to the HIV-1M/SIVcpz lineage. The strong sequence conservation of the TRIM19/PML sequences among primates suggests that this gene does not play a role in antiretroviral defense.

Suppression of short interfering RNA-mediated gene silencing by the structural proteins of hepatitis C virus.

Viruses have evolved strategies to overcome the antiviral effects of the host at different levels. Besides specific defence mechanisms, the host responds to viral infection via the interferon pathway and also by RNA interference (RNAi). However, several viruses have been identified that suppress RNAi. We addressed the question of whether hepatitis C virus (HCV) suppresses RNAi, using cell lines constitutively expressing green fluorescent protein (GFP) and inducibly expressing HCV proteins. It was found that short interfering RNA-mediated GFP gene silencing was inhibited when the entire HCV polyprotein was expressed. Further studies showed that HCV structural proteins, and in particular envelope protein 2 (E2), were responsible for this inhibition. Co-precipitation assays demonstrated that E2 bound to Argonaute-2 (Ago-2), a member of the RNA-induced silencing complex, RISC. Thus, HCV E2 that interacts with Ago-2 is able to suppress RNAi.

Modified SH2 domain to phototrap and identify phosphotyrosine proteins from subcellular sites within cells.

Spatial regulation of tyrosine phosphorylation is important for many aspects of cell biology. However, phosphotyrosine accounts for less than 1% of all phosphorylated substrates, and it is typically a very transient event in vivo. These factors complicate the identification of key tyrosine kinase substrates, especially in the context of their extraordinary spatial organization. Here, we describe an approach to identify tyrosine kinase substrates based on their subcellular distribution from within cells. This method uses an unnatural amino acid-modified Src homology 2 (SH2) domain that is expressed within cells and can covalently trap phosphotyrosine proteins on exposure to light. This SH2 domain-based photoprobe was targeted to cellular structures, such as the actin cytoskeleton, mitochondria, and cellular membranes, to capture tyrosine kinase substrates unique to each cellular region. We demonstrate that RhoA, one of the proteins associated with actin, can be phosphorylated on two tyrosine residues within the switch regions, suggesting that phosphorylation of these residues might modulate RhoA signaling to the actin cytoskeleton. We conclude that expression of SH2 domains within cellular compartments that are capable of covalent phototrapping can reveal the spatial organization of tyrosine kinase substrates that are likely to be important for the regulation of subcellular structures.

A-kinase anchoring proteins: scaffolding proteins in the heart.

The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network. Scaffolds that bind protein kinase A are called "A-kinase anchoring proteins" (AKAPs). In this review, we discuss recent advances in our understanding of how PKA is compartmentalized within the cardiac myocyte by AKAPs and how AKAP complexes modulate cardiac function in both health and disease.

Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis.

BACKGROUND: Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes. RESULTS: Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA), consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory. CONCLUSIONS: Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types.

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA from Bordetella pertussis.

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed.

Interaction of Fas(Apo-1/CD95) with proteins implicated in the ubiquitination pathway.

Fas(Apo-1/CD95), a receptor belonging to the tumor necrosis factor receptor family, induces apoptosis when triggered by Fas ligand. Upon its activation, the cytoplasmic domain of Fas binds several proteins which transmit the death signal. We used the yeast two-hybrid screen to isolate Fas-associated proteins. Here we report that the ubiquitin-conjugating enzyme UBC9 binds to Fas at the interface between the death domain and the membrane-proximal region of Fas. This interaction is also seen in vivo. UBC9 transiently expressed in HeLa cells bound to the co-expressed cytoplasmic segment of Fas. FAF1, a Fas-associated protein that potentiates apoptosis (Chu et al. (1996) Proc. Natl. Acad. Sci. USA 92, 11894-11898), was found to contain sequences similar to ubiquitin. These results suggest that proteins related to the ubiquitination pathway may modulate the Fas signaling pathway.

Biophysical characterization of tubulin tyrosine ligase-like proteins on microtubule dynamics

Report for the scientific sojourn carried out at the Cell Biology and Biophysics Unit from the National Institutes of Health, from 2010 to 2012.

Distributed all-atom simulations of intrinsically unstructured proteins

The Computational Biophysics Group at the Universitat Pompeu Fabra (GRIB-UPF) hosts two unique computational resources dedicated to the execution of large scale molecular dynamics (MD) simulations: (a) the ACMD molecular-dynamics software, used on standard personal computers with graphical processing units (GPUs); and (b) the GPUGRID. net computing network, supported by users distributed worldwide that volunteer GPUs for biomedical research. We leveraged these resources and developed studies, protocols and open-source software to elucidate energetics and pathways of a number of biomolecular systems, with a special focus on flexible proteins with many degrees of freedom. First, we characterized ion permeation through the bactericidal model protein Gramicidin A conducting one of the largest studies to date with the steered MD biasing methodology. Next, we addressed an open problem in structural biology, the determination of drug-protein association kinetics; we reconstructed the binding free energy, association, and dissaciociation rates of a drug like model system through a spatial decomposition and a Makov-chain analysis. The work was published in the Proceedings of the National Academy of Sciences and become one of the few landmark papers elucidating a ligand-binding pathway. Furthermore, we investigated the unstructured Kinase Inducible Domain (KID), a 28-peptide central to signalling and transcriptional response; the kinetics of this challenging system was modelled with a Markovian approach in collaboration with Frank Noe’s group at the Freie University of Berlin. The impact of the funding includes three peer-reviewed publication on high-impact journals; three more papers under review; four MD analysis components, released as open-source software; MD protocols; didactic material, and code for the hosting group.

Docking to heme proteins.

In silico screening has become a valuable tool in drug design, but some drug targets represent real challenges for docking algorithms. This is especially true for metalloproteins, whose interactions with ligands are difficult to parametrize. Our docking algorithm, EADock, is based on the CHARMM force field, which assures a physically sound scoring function and a good transferability to a wide range of systems, but also exhibits difficulties in case of some metalloproteins. Here, we consider the therapeutically important case of heme proteins featuring an iron core at the active site. Using a standard docking protocol, where the iron-ligand interaction is underestimated, we obtained a success rate of 28% for a test set of 50 heme-containing complexes with iron-ligand contact. By introducing Morse-like metal binding potentials (MMBP), which are fitted to reproduce density functional theory calculations, we are able to increase the success rate to 62%. The remaining failures are mainly due to specific ligand-water interactions in the X-ray structures. Testing of the MMBP on a second data set of non iron binders (14 cases) demonstrates that they do not introduce a spurious bias towards metal binding, which suggests that they may reliably be used also for cross-docking studies.

Splicing and the evolution of proteins in mammals.

It is often supposed that a protein's rate of evolution and its amino acid content are determined by the function and anatomy of the protein. Here we examine an alternative possibility, namely that the requirement to specify in the unprocessed RNA, in the vicinity of intron-exon boundaries, information necessary for removal of introns (e.g., exonic splice enhancers) affects both amino acid usage and rates of protein evolution. We find that the majority of amino acids show skewed usage near intron-exon boundaries, and that differences in the trends for the 2-fold and 4-fold blocks of both arginine and leucine show this to be owing to effects mediated at the nucleotide level. More specifically, there is a robust relationship between the extent to which an amino acid is preferred/avoided near boundaries and its enrichment/paucity in splice enhancers. As might then be expected, the rate of evolution is lowest near intron-exon boundaries, at least in part owing to splice enhancers, such that domains flanking intron-exon junctions evolve on average at under half the rate of exon centres from the same gene. In contrast, the rate of evolution of intronless retrogenes is highest near the domains where intron-exon junctions previously resided. The proportion of sequence near intron-exon boundaries is one of the stronger predictors of a protein's rate of evolution in mammals yet described. We conclude that after intron insertion selection favours modification of amino acid content near intron-exon junctions, so as to enable efficient intron removal, these changes then being subject to strong purifying selection even if nonoptimal for protein function. Thus there exists a strong force operating on protein evolution in mammals that is not explained directly in terms of the biology of the protein.

KnotProt: a database of proteins with knots and slipknots.

The protein topology database KnotProt, http://knotprot.cent.uw.edu.pl/, collects information about protein structures with open polypeptide chains forming knots or slipknots. The knotting complexity of the cataloged proteins is presented in the form of a matrix diagram that shows users the knot type of the entire polypeptide chain and of each of its subchains. The pattern visible in the matrix gives the knotting fingerprint of a given protein and permits users to determine, for example, the minimal length of the knotted regions (knot's core size) or the depth of a knot, i.e. how many amino acids can be removed from either end of the cataloged protein structure before converting it from a knot to a different type of knot. In addition, the database presents extensive information about the biological functions, families and fold types of proteins with non-trivial knotting. As an additional feature, the KnotProt database enables users to submit protein or polymer chains and generate their knotting fingerprints.

Impaired fast-spiking, suppressed cortical inhibition and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins

Voltage-gated K+ channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to −10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 −/− mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.