Structural basis for the regulation of L-type voltage-gated calcium channels: interactions between the N-terminal cytoplasmic domain and Ca2+-calmodulin

It is well-known that the opening of L-type voltage-gated calcium channels can be regulated by calmodulin (CaM). One of the main regulatory mechanisms is calcium-dependent inactivation (CDI), where binding of apo-CaM to the cytoplasmic C-terminal domain of the channel can effectively sense an increase in the local calcium ion concentration. Calcium-bound CaM can bind to the IQ-motif region of the C-terminal region and block the calcium channel, thereby providing a negative feedback mechanism that prevents the rise of cellular calcium concentrations over physiological limits. Recently, an additional Ca2+/CaM-binding motif (NSCaTE, N-terminal spatial Ca2+ transforming element) was identified in the amino terminal cytoplasmic region of Cav1.2 and Cav1.3. This motif exists only in Cav1.2 and Cav1.3 channels, and a pronounced N-lobe (Ca2+/CaM) CDI effect was found for Cav1.3. To understand the molecular basis of this interaction, the complexes of Ca2+/CaM with the biosynthetically produced N-terminal region (residues 1–68) and NSCaTE peptide (residues 48–68) were investigated. We discovered that the NSCaTE motif in the N-terminal cytoplasmic region adopts an α-helical conformation, most likely due to its high alanine content. Additionally, the complex exhibits an unusual 1:2 protein:peptide stoichiometry when bound to Ca2+-CaM, and the N-lobe of CaM has a much stronger affinity for the peptide than the C-lobe. The complex structures of the isolated N- and C-lobe of Ca2+/CaM and the NSCaTE peptide were determined by nuclear magnetic resonance spectroscopy and data-driven protein-docking methods. Moreover, we also demonstrated that calcium binding protein 1, which competes with CaM for binding to the C-terminal cytoplasmic domain, binds only weakly to the NSCaTE region. The structures provide insights into the possible roles of this motif in the calcium regulatory network. Our study provides structural evidence for the CaM-bridge model proposed in previous studies

Metabolomics: is it useful for inflammatory bowel diseases?

Purpose of Review: The assessment of metabolite profiles in biofluids has become a powerful method for the detection of biomarker molecules and disease mechanisms. This review outlines the recent advances in the use of metabolomic techniques to study inflammatory bowel diseases (IBDs). Recent findingsThe last few years have seen an increase in the studies of experimental and human IBD focusing on the search for small metabolites, such as amino acids, bases, and tricarboxylic acid cycle intermediates. Experimental methods for the screening of metabolites in serum, urine, fecal extracts, and colon tissue include H-1 NMR spectroscopy, gas chromatography-mass spectrometry, and liquid chromatography methods. Several studies demonstrate that IBD patients and healthy individuals, as well as the IBD subtypes, can be distinguished using metabolic profiling. Metabolomic data of fecal extracts and urine have revealed disruptions in bacterial populations, findings that are indicative of a possible involvement of the microbiome in the development of IBDs. SummaryMetabolites from biofluids can be detected in IBDs by different experimental methods that allow successful separation of IBD subtypes from healthy cohorts. Knowledge of a unique metabolomic fingerprint in IBDs could be useful for diagnosis, treatment, and detection of disease mechanisms

Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action

AbstractAntimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation–π interactions, thereby facilitating enhanced peptide–membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular α-helices and β-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented

Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures

<p>Abstract</p> <p>Background</p> <p>Opium poppy (<it>Papaver somniferum</it>) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (¹H NMR) metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor.</p> <p>Results</p> <p>Metabolite fingerprinting and compound-specific profiling showed the extensive reprogramming of primary metabolic pathways in association with the induction of alkaloid biosynthesis in response to elicitor treatment. Using Chenomx NMR Suite v. 4.6, a software package capable of identifying and quantifying individual compounds based on their respective signature spectra, the levels of 42 diverse metabolites were monitored over a 100-hour time course in control and elicitor-treated opium poppy cell cultures. Overall, detectable and dynamic changes in the metabolome of elicitor-treated cells, especially in cellular pools of carbohydrates, organic acids and non-protein amino acids were detected within 5 hours after elicitor treatment. The metabolome of control cultures also showed substantial modulations 80 hours after the start of the time course, particularly in the levels of amino acids and phospholipid pathway intermediates. Specific flux modulations were detected throughout primary metabolism, including glycolysis, the tricarboxylic acid cycle, nitrogen assimilation, phospholipid/fatty acid synthesis and the shikimate pathway, all of which generate secondary metabolic precursors.</p> <p>Conclusion</p> <p>The response of cell cultures to elicitor treatment involves the extensive reprogramming of primary and secondary metabolism, and associated cofactor biosynthetic pathways. A high-resolution map of the extensive reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures is provided.</p

Adolescents and MP3 Players: Too Many Risks, Too Few Precautions

OBJECTIVE. The goal was to assess risky and protective listening behaviors of adolescent users of MP3 players and the association of these behaviors with demographic characteristics and frequency of use. METHODS. In 2007, 1687 adolescents (12–19 years of age) in 68 classes in 15 Dutch secondary schools were invited to complete questionnaires about their music-listening behaviors. RESULTS. Ninety percent of participants reported listening to music through earphones on MP3 players; 32.8% were frequent users, 48.0% used high volume settings, and only 6.8% always or nearly always used a noise-limiter. Frequent users were _4 times more likely to listen to high-volume music than were infrequent users, and adolescents in practical prevocational schools were more than twice as likely to listen to high-volume music as were those attending preuniversity education. CONCLUSIONS. When using MP3 players, adolescents are very likely to engage in risky listening behaviors and are unlikely to seek protection. Frequent MP3 player use is an indicator of other risky listening behaviors, such as listening at high volumes and failing to use noise-limiter

Metabolomics: is it useful for inflammatory bowel diseases?

Purpose of Review: The assessment of metabolite profiles in biofluids has become a powerful method for the detection of biomarker molecules and disease mechanisms. This review outlines the recent advances in the use of metabolomic techniques to study inflammatory bowel diseases (IBDs). Recent findingsThe last few years have seen an increase in the studies of experimental and human IBD focusing on the search for small metabolites, such as amino acids, bases, and tricarboxylic acid cycle intermediates. Experimental methods for the screening of metabolites in serum, urine, fecal extracts, and colon tissue include H-1 NMR spectroscopy, gas chromatography-mass spectrometry, and liquid chromatography methods. Several studies demonstrate that IBD patients and healthy individuals, as well as the IBD subtypes, can be distinguished using metabolic profiling. Metabolomic data of fecal extracts and urine have revealed disruptions in bacterial populations, findings that are indicative of a possible involvement of the microbiome in the development of IBDs. SummaryMetabolites from biofluids can be detected in IBDs by different experimental methods that allow successful separation of IBD subtypes from healthy cohorts. Knowledge of a unique metabolomic fingerprint in IBDs could be useful for diagnosis, treatment, and detection of disease mechanisms

The Solution Structure, Binding Properties, and Dynamics of the Bacterial Siderophore-binding Protein FepB

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large "Venus flytrap" conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225-250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs

The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles

AbstractLactoferricin B (LfcinB) is a 25-residue antimicrobial peptide released from bovine lactoferrin upon pepsin digestion. The antimicrobial center of LfcinB consists of six residues (RRWQWR-NH2), and it possesses similar bactericidal activity to LfcinB. The structure of the six-residue peptide bound to sodium dodecyl sulfate (SDS) micelles has been determined by NMR spectroscopy and molecular dynamics refinement. The peptide adopts a well defined amphipathic structure when bound to SDS micelles with the Trp sidechains separated from the Arg residues. Additional evidence demonstrates that the peptide is oriented in the micelle such that the Trp residues are more deeply buried in the micelle than the Arg and Gln residues

Structure–function studies of chemokine-derived carboxy-terminal antimicrobial peptides

AbstractRecent reports which show that several chemokines can act as direct microbicidal agents have drawn renewed attention to these chemotactic signalling proteins. Here we present a structure–function analysis of peptides derived from the human chemokines macrophage inflammatory protein-3α (MIP-3α/CCL20), interleukin-8 (IL-8), neutrophil activating protein-2 (NAP-2) and thrombocidin-1 (TC-1). These peptides encompass the C-terminal α-helices of these chemokines, which have been suggested to be important for the direct antimicrobial activities. Far-UV CD spectroscopy showed that the peptides are unstructured in aqueous solution and that a membrane mimetic solvent is required to induce a helical secondary structure. A co-solvent mixture was used to determine solution structures of the peptides by two-dimensional 1H-NMR spectroscopy. The highly cationic peptide, MIP-3α51–70, had the most pronounced antimicrobial activity and displayed an amphipathic structure. A shorter version of this peptide, MIP-3α59–70, remained antimicrobial but its structure and mechanism of action were unlike that of the former peptide. The NAP-2 and TC-1 proteins differ in their sequences only by the deletion of two C-terminal residues in TC-1, but intact TC-1 is a very potent antimicrobial while NAP-2 is inactive. The corresponding C-terminal peptides, NAP-250–70 and TC-150–68, had very limited and no bactericidal activity, respectively. This suggests that other regions of TC-1 contribute to its bactericidal activity. Altogether, this work provides a rational structural basis for the biological activities of these peptides and proteins and highlights the importance of experimental characterization of peptide fragments as distinct entities because their activities and structural properties may differ substantially from their parent proteins

Characterization and prediction of the mechanism of action of antibiotics through NMR metabolomics

Background: The emergence of antibiotic resistant pathogenic bacteria has reduced our ability to combat infectious diseases. At the same time the numbers of new antibiotics reaching the market have decreased. This situation has created an urgent need to discover novel antibiotic scaffolds. Recently, the application of pattern recognition techniques to identify molecular fingerprints in ‘omics’ studies, has emerged as an important tool in biomedical research and laboratory medicine to identify pathogens, to monitor therapeutic treatments or to develop drugs with improved metabolic stability, toxicological profile and efficacy. Here, we hypothesize that a combination of metabolic intracellular fingerprints and extracellular footprints would provide a more comprehensive picture about the mechanism of action of novel antibiotics in drug discovery programs. Results: In an attempt to integrate the metabolomics approach as a classification tool in the drug discovery processes, we have used quantitative 1H NMR spectroscopy to study the metabolic response of Escherichia coli cultures to different antibiotics. Within the frame of our study the effects of five different and well-known antibiotic classes on the bacterial metabolome were investigated both by intracellular fingerprint and extracellular footprint analysis. The metabolic fingerprints and footprints of bacterial cultures were affected in a distinct manner and provided complementary information regarding intracellular and extracellular targets such as protein synthesis, DNA and cell wall. While cell cultures affected by antibiotics that act on intracellular targets showed class-specific fingerprints, the metabolic footprints differed significantly only when antibiotics that target the cell wall were applied. In addition, using a training set of E. coli fingerprints extracted after treatment with different antibiotic classes, the mode of action of streptomycin, tetracycline and carbenicillin could be correctly predicted. Conclusion: The metabolic profiles of E. coli treated with antibiotics with intracellular and extracellular targets could be separated in fingerprint and footprint analysis, respectively and provided complementary information. Based on the specific fingerprints obtained for different classes of antibiotics, the mode of action of several antibiotics could be predicted. The same classification approach should be applicable to studies of other pathogenic bacteria