Molecular dynamic simulations reveal the structural determinants of Fatty Acid binding to oxy-myoglobin.

The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a "U" shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state.

TRPV channel-mediated calcium transients in nociceptor neurons are dispensable for avoidance behaviour.

Animals need to sense and react to potentially dangerous environments. TRP ion channels participate in nociception, presumably via Ca(2+) influx, in most animal species. However, the relationship between ion permeation and animals' nocifensive behaviour is unknown. Here we use an invertebrate animal model with relevance for mammalian pain. We analyse the putative selectivity filter of OSM-9, a TRPV channel, in osmotic avoidance behaviour of Caenorhabditis elegans. Using mutagenized OSM-9 expressed in the head nociceptor neuron, ASH, we study nocifensive behaviour and Ca(2+) influx. Within the selectivity filter, M(601)-F(609), Y604G strongly reduces avoidance behaviour and eliminates Ca(2+) transients. Y604F also abolishes Ca(2+) transients in ASH, while sustaining avoidance behaviour, yet it disrupts behavioral plasticity. Homology modelling of the OSM-9 pore suggests that Y(604) may assume a scaffolding role. Thus, aromatic residues in the OSM-9 selectivity filter are critical for pain behaviour and ion permeation. These findings have relevance for understanding evolutionary roots of mammalian nociception.

Towards neuronal organoids: a method for long-term culturing of high-density hippocampal neurons.

One of the goals in neuroscience is to obtain tractable laboratory cultures that closely recapitulate in vivo systems while still providing ease of use in the lab. Because neurons can exist in the body over a lifetime, long-term culture systems are necessary so as to closely mimic the physiological conditions under laboratory culture conditions. Ideally, such a neuronal organoid culture would contain multiple cell types, be highly differentiated, and have a high density of interconnected cells. However, before these types of cultures can be created, certain problems associated with long-term neuronal culturing must be addressed. We sought to develop a new protocol which may further prolong the duration and integrity of E18 rat hippocampal cultures. We have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes-particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons.

Reevaluation of the evolutionary events within recA/RAD51 phylogeny.

BACKGROUND: The recA/RAD51 gene family encodes a diverse set of recombinase proteins that affect homologous recombination, DNA-repair, and genome stability. The recA gene family is expressed across all three domains of life - Eubacteria, Archaea, and Eukaryotes - and even in some viruses. To date, efforts to resolve the deep evolutionary origins of this ancient protein family have been hindered by the high sequence divergence between paralogous groups (i.e. ~30% average pairwise identity). RESULTS: Through large taxon sampling and the use of a phylogenetic algorithm designed for inferring evolutionary events in highly divergent paralogs, we obtained a robust, parsimonious and more refined phylogenetic history of the recA/RAD51 superfamily. CONCLUSIONS: In summary, our model for the evolution of recA/RAD51 family provides a better understanding of the ancient origin of recA proteins and the multiple events that lead to the diversification of recA homologs in eukaryotes, including the discovery of additional RAD51 sub-families.

PHYRN: a robust method for phylogenetic analysis of highly divergent sequences.

Both multiple sequence alignment and phylogenetic analysis are problematic in the "twilight zone" of sequence similarity (≤ 25% amino acid identity). Herein we explore the accuracy of phylogenetic inference at extreme sequence divergence using a variety of simulated data sets. We evaluate four leading multiple sequence alignment (MSA) methods (MAFFT, T-COFFEE, CLUSTAL, and MUSCLE) and six commonly used programs of tree estimation (Distance-based: Neighbor-Joining; Character-based: PhyML, RAxML, GARLI, Maximum Parsimony, and Bayesian) against a novel MSA-independent method (PHYRN) described here. Strikingly, at "midnight zone" genetic distances (~7% pairwise identity and 4.0 gaps per position), PHYRN returns high-resolution phylogenies that outperform traditional approaches. We reason this is due to PHRYN's capability to amplify informative positions, even at the most extreme levels of sequence divergence. We also assess the applicability of the PHYRN algorithm for inferring deep evolutionary relationships in the divergent DANGER protein superfamily, for which PHYRN infers a more robust tree compared to MSA-based approaches. Taken together, these results demonstrate that PHYRN represents a powerful mechanism for mapping uncharted frontiers in highly divergent protein sequence data sets.

Predicting protein folds with fold-specific PSSM libraries.

Accurately assigning folds for divergent protein sequences is a major obstacle to structural studies. Herein, we outline an effective method for fold recognition using sets of PSSMs, each of which is constructed for different protein folds. Our analyses demonstrate that FSL (Fold-specific Position Specific Scoring Matrix Libraries) can predict/relate structures given only their amino acid sequences of highly divergent proteins. This ability to detect distant relationships is dependent on low-identity sequence alignments obtained from FSL. Results from our experiments demonstrate that FSL perform well in recognizing folds from the "twilight-zone" SABmark dataset. Further, this method is capable of accurate fold prediction in newly determined structures. We suggest that by building complete PSSM libraries for all unique folds within the Protein Database (PDB), FSL can be used to rapidly and reliably annotate a large subset of protein folds at proteomic level. The related programs and fold-specific PSSMs for our FSL are publicly available at: http://ccp.psu.edu/download/FSLv1.0/.

Conserved GXXXG- and S/T-like motifs in the transmembrane domains of NS4B protein are required for hepatitis C virus replication.

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra- and intermolecular interactions required for NS4B structure/function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle.

TRPC channels in pheromone sensing.

Pheromone recognition relies on an amplification cascade that is triggered by pheromone binding to G protein-coupled receptors (GPCR). The first step in translation of GPCR activation by pheromones in the vomeronasal organ and main olfactory epithelium (MOE) into a cellular response is the activation of a transient receptor potential (TRP) family member, TRPC2 [Zufall, F., Ukhanov, K., Lucas, P., Liman, E. R., and Leinders-Zufall, T. (2005). Neurobiology of TRPC2: From gene to behavior. Pflugers Arch.451, 61-71; Yildirim, E., and Birnbaumer, L. (2007). TRPC2: Molecular biology and functional importance. Handb. Exp. Pharmacol. 53-75]. The members of the canonical (TRPC) family of TRP channels mediate membrane permeability, specifically, Ca(2+) influx into the cytoplasm in response to activation of GPCR and tyrosine kinase receptors by hormones, neurotransmitters, and growth factors [Nilius, B. (2007). TRP channels in disease. Biochim. Biophys. Acta1772, 805-812; Venkatachalam, K., and Montell, C. (2007). TRP channels. Annu. Rev. Biochem.76, 387-417]. Mechanisms of their activation have been the focus of intense interest during the last decade. The data obtained from studies of TRPC2 have resulted in a better understanding of ion channel physiology and led to novel paradigms in modern cell biology [Lucas, P., Ukhanov, K., Leinders-Zufall, T., and Zufall, F. (2003). A diacylglycerol-gated cation channel in vomeronasal neuron dendrites is impaired in TRPC2 mutant mice: Mechanism of pheromone transduction. Neuron40, 551-561; Stowers, L., Holy, T. E., Meister, M., Dulac, C., and Koentges, G. (2002). Loss of sex discrimination and male-male aggression in mice deficient for TRP2. Science295, 1493-1500; Leypold, B. G., Yu, C. R., Leinders-Zufall, T., Kim, M. M., Zufall, F., and Axel, R. (2002). Altered sexual and social behaviors in trp2 mutant mice. Proc. Natl. Acad. Sci. USA99, 6376-6381]. Although TRPC2 activation by pheromones presents one of the most straightforward examples of physiological function of TRPC channels, the molecular aspects of its activation are not well understood (Yildirim, E., and Birnbaumer, L. (2007). TRPC2: Molecular biology and functional importance. Handb. Exp. Pharmacol. 53-75). It is natural to expect that better understanding of TRPC2 activation mechanisms will lead to breakthroughs in understanding ion channel activation mechanisms, as well as applied behavioral pharmacology. The present review is focused on the current knowledge of TRPC2 physiology with a specific focus on TRPC activation mechanisms.

Death-associated protein kinase-mediated cell death modulated by interaction with DANGER.

Death-associated protein kinase (DAPK) is a key player in multiple cell death signaling pathways. We report that DAPK is regulated by DANGER, a partial MAB-21 domain-containing protein. DANGER binds directly to DAPK and inhibits DAPK catalytic activity. DANGER-deficient mouse embryonic fibroblasts and neurons exhibit greater DAPK activity and increased sensitivity to cell death stimuli than do wild-type control cells. In addition, DANGER-deficient mice manifest more severe brain damage after acute excitotoxicity and transient cerebral ischemia than do control mice. Accordingly, DANGER may physiologically regulate the viability of neurons and represent a potential therapeutic target for stroke and neurodegenerative diseases.

Phylogenetic Profiles Reveal Structural and Functional Determinants of Lipid-binding.

One of the major challenges in the genomic era is annotating structure/function to the vast quantities of sequence information now available. Indeed, most of the protein sequence database lacks comprehensive annotation, even when experimental evidence exists. Further, within structurally resolved and functionally annotated protein domains, additional functionalities contained in these domains are not apparent. To add further complication, small changes in the amino-acid sequence can lead to profound changes in both structure and function, underscoring the need for rapid and reliable methods to analyze these types of data. Phylogenetic profiles provide a quantitative method that can relate the structural and functional properties of proteins, as well as their evolutionary relationships. Using all of the structurally resolved Src-Homology-2 (SH2) domains, we demonstrate that knowledge-bases can be used to create single-amino acid phylogenetic profiles which reliably annotate lipid-binding. Indeed, these measures isolate the known phosphotyrosine and hydrophobic pockets as integral to lipid-binding function. In addition, we determined that the SH2 domain of Tec family kinases bind to lipids with varying affinity and specificity. Simulating mutations in Bruton's tyrosine kinase (BTK) that cause X-Linked Agammaglobulinemia (XLA) predict that these mutations alter lipid-binding, which we confirm experimentally. In light of these results, we propose that XLA-causing mutations in the SH3-SH2 domain of BTK alter lipid-binding, which could play a causative role in the XLA-phenotype. Overall, our study suggests that the number of lipid-binding proteins is drastically underestimated and, with further development, phylogenetic profiles can provide a method for rapidly increasing the functional annotation of protein sequences.

Phylogenetic profiles reveal structural/functional determinants of TRPC3 signal-sensing antennae.

Biochemical assessment of channel structure/function is incredibly challenging. Developing computational tools that provide these data would enable translational research, accelerating mechanistic experimentation for the bench scientist studying ion channels. Starting with the premise that protein sequence encodes information about structure, function and evolution (SF&E), we developed a unified framework for inferring SF&E from sequence information using a knowledge-based approach. The Gestalt Domain Detection Algorithm-Basic Local Alignment Tool (GDDA-BLAST) provides phylogenetic profiles that can model, ab initio, SF&E relationships of biological sequences at the whole protein, single domain and single-amino acid level.1,2 In our recent paper,4 we have applied GDDA-BLAST analysis to study canonical TRP (TRPC) channels1 and empirically validated predicted lipid-binding and trafficking activities contained within the TRPC3 TRP_2 domain of unknown function. Overall, our in silico, in vitro, and in vivo experiments support a model in which TRPC3 has signal-sensing antennae which are adorned with lipid-binding, trafficking and calmodulin regulatory domains. In this Addendum, we correlate our functional domain analysis with the cryo-EM structure of TRPC3.3 In addition, we synthesize recent studies with our new findings to provide a refined model on the mechanism(s) of TRPC3 activation/deactivation.

Phospholipase C-gamma binds directly to the Na+/H+ exchanger 3 and is required for calcium regulation of exchange activity.

Multiple studies suggest that phospholipase C-gamma (PLC-gamma) contributes to regulation of sodium/hydrogen exchanger 3 (NHE3) in the small intestine, although the mechanism(s) for this regulation remain unknown. We demonstrate here that PLC-gamma binds directly to the C terminus of NHE3 and exists in similar sized multiprotein complexes as NHE3. This binding is dynamic and decreases with elevated [Ca(2+)](i). The PLC-gamma-binding site in NHE3 was identified (amino acids 586-605) and shown to be a critical regulatory domain for protein complex formation, because when it is mutated, NHE3 binding to PLC-gamma as well as NHERF2 is lost. An inhibitory peptide, which binds to the Src homology 2 domains contained in PLC-gamma without interrupting binding of PLC-gamma to NHE3, was used to probe a non-lipase-dependent role of PLC-gamma. In the presence of this peptide, carbachol-stimulated calcium inhibition of NHE3 was lost. These results mirror previous studies with the transient receptor potential channel and suggest that PLC-gamma may play a common role in regulating the cell-surface expression of ion transporters.

PKC and PLA2: probing the complexities of the calcium network.

Lipid signaling and phosphorylation cascades are fundamental to calcium signaling networks. In this review, we will discuss the recent laboratory findings for the phospholipase A(2) (PLA(2))/protein kinase C (PKC) pathway within cellular calcium networks. The complexity and connectivity of these ubiquitous cellular signals make interpretation of experimental results extremely challenging. We present here computational methods which have been developed to conquer such complex data, and how they can be used to make models capable of accurately predicting cellular responses within multiple calcium signaling pathways. We propose that information obtained from network analysis and computational techniques provides a rich source of knowledge which can be directly translated to the laboratory benchtop.

The integrative function of TRPC channels.

TRPC is a subfamily of Transient Receptor Potential channels that have the highest degree of homology to the Drosophila photoreceptors' TRP. TRPC open in response to stimulation of plasma membrane receptors that activate phospholipase C, triggering transmembrane Ca2+ influx. TRPC activity has been directly implicated in regulation of vascular tone, kidney filtration, acrosomal reaction and pheromone recognition. As humans contain six TRPC channels, which form homo- and hetero-tetramers, TRPCs are capable of forming multiple channels of varying current/voltage relationships and activation properties. This allows TRPC to participate in an array of intercellular pathways induced by chemical mediators including hormones, neurotransmitters and growth factors. The strength of TRPC response to stimulation is modulated by several factors such as covalent modification, interaction with auxiliary proteins and changes in the lipid environment. The existence of several modulatory inputs that converge on TRPC enables integration of various stimuli and differentiation of Ca2+ signaling in specific tissues. This synthesizes the current literature describing the known functions and phenomenology associated with TRPC channels, with a specific focus on the activation and modulatory mechanisms. We suggest that the polymodal regulation of TRPC channels is likely to explain many specific aspects of TRPC behavior in different tissues.

Glutamatergic regulation of serine racemase via reversal of PIP2 inhibition.

D-serine is a physiologic coagonist with glutamate at NMDA-subtype glutamate receptors. As D-serine is localized in glia, synaptically released glutamate presumably stimulates the glia to form and release D-serine, enabling glutamate/D-serine cotransmission. We show that serine racemase (SR), which generates D-serine from L-serine, is physiologically inhibited by phosphatidylinositol (4,5)-bisphosphate (PIP2) presence in membranes where SR is localized. Activation of metabotropic glutamate receptors (mGluR5) on glia leads to phospholipase C-mediated degradation of PIP2, relieving SR inhibition. Thus mutants of SR that cannot bind PIP2 lose their membrane localizations and display a 4-fold enhancement of catalytic activity. Moreover, mGluR5 activation of SR activity is abolished by inhibiting phospholipase C.

TRP_2, a lipid/trafficking domain that mediates diacylglycerol-induced vesicle fusion.

We recently modeled transient receptor potential (TRP) channels using the Gestalt Domain Detection Algorithm-Basic Local Alignment Tool (GDDA-BLAST), which derives structural, functional, and evolutionary information from primary amino acid sequences using phylogenetic profiles ( Ko, K. D., Hong, Y., Chang, G. S., Bhardwaj, G., van Rossum, D. B., and Patterson, R. L. (2008) Physics Arch. Quant. Methods arXiv: 0806.2394v1 ). Herein we test our functional predictions for the TRP_2 domain of TRPC3; a domain of unknown function that is conserved in all TRPC channels. Our functional models of this domain identify both lipid binding and trafficking activities. In this study, we reveal: (i) a novel structural determinant of ion channel sensitivity to lipids, (ii) a molecular mechanism for the difference between diacylglycerol (DAG)-sensitive and DAG-insensitive TRPC subfamilies, and (iii) evidence that TRPC3 can comprise part of the vesicle fusion machinery. Indeed, the TRPC3 TRP_2 domain mediates channel trafficking to the plasma membrane and binds to plasma membrane lipids. Further, mutations in TRP_2, which alter lipid binding, also disrupt the DAG-mediated fusion of TRPC3-containing vesicles with the plasma membrane without disrupting SNARE interactions. Importantly, these data agree with the known role of DAG in membrane destabilization, which facilitates SNARE-dependent synaptic vesicle fusion ( Villar, A. V., Goni, F. M., and Alonso, A. (2001) FEBS Lett. 494, 117-120 and Goni, F. M., and Alonso, A. (1999) Prog. Lipid Res. 38, 1-48 ). Taken together, functional models generated by GDDA-BLAST provide a computational platform for deriving domain functionality, which can have in vivo and mechanistic relevance.

Phylogenetic profiles reveal evolutionary relationships within the "twilight zone" of sequence similarity.

Inferring evolutionary relationships among highly divergent protein sequences is a daunting task. In particular, when pairwise sequence alignments between protein sequences fall <25% identity, the phylogenetic relationships among sequences cannot be estimated with statistical certainty. Here, we show that phylogenetic profiles generated with the Gestalt Domain Detection Algorithm-Basic Local Alignment Tool (GDDA-BLAST) are capable of deriving, ab initio, phylogenetic relationships for highly divergent proteins in a quantifiable and robust manner. Notably, the results from our computational case study of the highly divergent family of retroelements accord with previous estimates of their evolutionary relationships. Taken together, these data demonstrate that GDDA-BLAST provides an independent and powerful measure of evolutionary relationships that does not rely on potentially subjective sequence alignment. We demonstrate that evolutionary relationships can be measured with phylogenetic profiles, and therefore propose that these measurements can provide key insights into relationships among distantly related and/or rapidly evolving proteins.

HSP90 regulates cell survival via inositol hexakisphosphate kinase-2.

Heat-shock proteins (HSPs) are abundant, inducible proteins best known for their ability to maintain the conformation of proteins and to refold damaged proteins. Some HSPs, especially HSP90, can be antiapoptotic and the targets of anticancer drugs. Inositol hexakisphosphate kinase-2 (IP6K2), one of a family of enzymes generating the inositol pyrophosphate IP7 [diphosphoinositol pentakisphosphate (5-PP-IP5)], mediates apoptosis. Increased IP6K2 activity sensitizes cancer cells to stressors, whereas its depletion blocks cell death. We now show that HSP90 physiologically binds IP6K2 and inhibits its catalytic activity. Drugs and selective mutations that abolish HSP90-IP6K2 binding elicit activation of IP6K2, leading to cell death. Thus, the prosurvival actions of HSP90 reflect the inhibition of IP6K2, suggesting that selectively blocking this interaction could provide effective and safer modes of chemotherapy.

Ancient origin of the new developmental superfamily DANGER.

Developmental proteins play a pivotal role in the origin of animal complexity and diversity. We report here the identification of a highly divergent developmental protein superfamily (DANGER), which originated before the emergence of animals (approximately 850 million years ago) and experienced major expansion-contraction events during metazoan evolution. Sequence analysis demonstrates that DANGER proteins diverged via multiple mechanisms, including amino acid substitution, intron gain and/or loss, and recombination. Divergence for DANGER proteins is substantially greater than for the prototypic member of the superfamily (Mab-21 family) and other developmental protein families (e.g., WNT proteins). DANGER proteins are widely expressed and display species-dependent tissue expression patterns, with many members having roles in development. DANGER1A, which regulates the inositol trisphosphate receptor, promotes the differentiation and outgrowth of neuronal processes. Regulation of development may be a universal function of DANGER family members. This family provides a model system to investigate how rapid protein divergence contributes to morphological complexity.

Action of TFII-I outside the nucleus as an inhibitor of agonist-induced calcium entry.

TFII-I is a transcription factor and a target of phosphorylation by Bruton's tyrosine kinase. In humans, deletions spanning the TFII-I locus are associated with a cognitive defect, the Williams-Beuren cognitive profile. We report an unanticipated role of TFII-I outside the nucleus as a negative regulator of agonist-induced calcium entry (ACE) that suppresses surface accumulation of TRPC3 (transient receptor potential C3) channels. Inhibition of ACE by TFII-I requires phosphotyrosine residues that engage the SH2 (Src-homology 2) domains of phospholipase C-g (PLC-g) and an interrupted, pleckstrin homology (PH)-like domain that binds the split PH domain of PLC-g. Our observations suggest a model in which TFII-I suppresses ACE by competing with TRPC3 for binding to PLC-g.