High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.     

Protein identification and tracking in two-dimensional electrophoretic gels by minimal protein identifiers

Protein identification by matrix-assisted laser desorption/ionization mass-spectrometry peptide mass fingerprinting (MALDI-MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low-molecular-mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI-MS-based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two-dimensional gel electrophoresis (2-DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI-MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein-specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF-Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2-DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.     

Heterocyclic analogues of squamocin as inhibitors of mitochondrial complex I. On the role of the terminal lactone of annonaceous acetogenins

Heterocyclic analogues of squamocin have been semisynthesized by condensation reactions between squamocin-derived alpha-keto esters and heterodinucleophiles. The strong complex I inhibitory potency of squamocin-benzimidazole, a hybrid derivative, illustrates for the first time the functional analogy existing between the terminal butenolide of annonaceous acetogenins and heteroaromatic substructures of classic inhibitors of the enzyme. This finding supports the categorization of this atypical group of inhibitors as antagonists of the ubiquinone substrates. In addition, competition experiments of squamocin-benzimidazole versus squamocin and rolliniastatin-2 suggest that the binding of this hybrid inhibitor is responsible for a negative allosteric effect at the level of the first ubiquinone-binding site (A site) of mitochondrial complex I. This result supports the existence of a large cooperatively regulated inhibitor/ubiquinone-binding pocket located within the catalytic core of the enzyme, consisting of the association of the previously defined affinity sites A and B.     

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.     

Pneumococcal Carriage in Children under Five Years in Uganda-Will Present Pneumococcal Conjugate Vaccines Be Appropriate?

BackgroundPneumonia is the major cause of death in children globally, with more than 900,000 deaths annually in children under five years of age. Streptococcus pneumoniae causes most deaths, most often in the form of community acquired pneumonia. Pneumococcal conjugate vaccines (PCVs) are currently being implemented in many low-income countries. PCVs decrease vaccine-type pneumococcal carriage, a prerequisite for invasive pneumococcal disease, and thereby affects pneumococcal disease and transmission. In Uganda, PCV was launched in 2014, but baseline data is lacking for pneumococcal serotypes in carriage.ObjectivesTo study pneumococcal nasopharyngeal carriage and serotype distribution in children under 5 years of age prior to PCV introduction in UgandaMethodsThree cross-sectional pneumococcal carriage surveys were conducted in 2008, 2009 and 2011, comprising respectively 150, 587 and 1024 randomly selected children aged less than five years from the Iganga/Mayuge Health and Demographic Surveillance Site. The caretakers were interviewed about illness history of the child and 1723 nasopharyngeal specimens were collected. From these, 927 isolates of S. pneumoniae were serotyped.ResultsOverall, the carriage rate of S. pneumoniae was 56% (957/1723). Pneumococcal carriage was associated with illness on the day of the interview (OR = 1.50, p = 0.04). The most common pneumococcal serotypes were in descending order 19F (16%), 23F (9%), 6A (8%), 29 (7%) and 6B (7%). One percent of the strains were non-typeable. The potential serotype coverage rate for PCV10 was 42% and 54% for PCV13.ConclusionAbout half of circulating pneumococcal serotypes in carriage in the Ugandan under-five population studied was covered by available PCVs.     

Synthesis and biological evaluation of 3'-carboranyl thymidine analogues

Boron neutron capture therapy (BNCT) is a chemoradio-therapeutic method for the treatment of cancer. It depends on the selective targeting of tumor cells by boron-containing compounds. One category of BNCT agents with potential to selectively target tumor cells may be thymidine derivatives substituted at the 3'-position with appropriate boron moieties. Thus, several thymidine analogues were synthesized with a carborane cluster bound to the 3'-position either through an ether or a carbon linkage. The latter are the first reported carborane-containing nucleosides in which the carboranyl entity is directly linked to the carbohydrate portion of the nucleoside by a carbon-carbon bond. Low but significant phosphorylation rates in the range of 0.18% that of thymidine were observed for the carbon-linked 3'-carboranyl thymidine analogues in phosphoryl transfer assays using recombinant preparations of thymidine kinases 1 (TK1) and thymidine kinases 2 (TK2). Some of the ether-linked 3'-carboranyl thymidine analogues appeared to be slightly unstable under acidic as well as phosphoryl transfer assay conditions and were, if at all, poor substrates for TK1.