Methoxyoxalamido chemistry in the synthesis of novel amino linker and spacer phosphoramidites: a robust means for stability, structural versatility, and optimal tether length

We have developed a general route to the synthesis of novel amino linker and spacer phosphoramidites utilizing methoxyoxalamido (MOX) chemistry. The synthesis makes use of readily available and inexpensive primary aliphatic amino alcohols and diamines to produce a rich and diverse variety of phosphoramidites. Among these are monomers with exceptionally long (up to 56 atoms in length) amphipathic tethering arms. The chemistry bestows exceptional control over the physical characteristics within the tethers through the selection of appropriate building blocks. Furthermore, MOX chemistry enables fairly rapid assembly of these discrete-length tethers in a modular fashion. All novel phosphoramidites were successfully used in automated syntheses of 5'-modified oligonucleotides.     

Rhodopsin determinants for transducin activation: a gain-of-function approach

Three cytoplasmic loops in the G protein-coupled receptor rhodopsin, C2, C3, and C4, have been implicated as key sites for binding and activation of the visual G protein transducin. Non-helical portions of the C2- and C3-loops and the cytoplasmic helix-8 from the C4 loop were targeted for a "gain-of-function" mutagenesis to identify rhodopsin residues critical for transducin activation. Mutant opsins with residues 140-148 (C2-loop), 229-244 (C3-loop), or 310-320 (C4-loop) substituted by poly-Ala sequences of equivalent lengths served as templates for mutagenesis. The template mutants with poly-Ala substitutions in the C2- and C3-loops formed the 500-nm absorbing pigments but failed to activate transducin. Reverse substitutions of the Ala residues by rhodopsin residues have been generated in each of the templates. Significant ( approximately 50%) restoration of the rhodopsin/transducin coupling was achieved with re-introduction of residues Cys140/Lys141 and Arg147/Phe148 into the C2 template. The reverse substitutions of the C3-loop residues Thr229/Val230 and Ser240/Thr242/Thr243/Gln244 produced a pigment with a full capacity for transducin activation. The C4 template mutant was unable to bind 11-cis-retinal, and the presence of Asn310/Lys311 was required for correct folding of the protein. Subsequent mutagenesis of the C4-loop revealed the role of Phe313 and Met317. On the background of Asn310/Lys311, the inclusion of Phe313 and Met317 produced a mutant pigment with the potency of transducin activation equal to that of the wild-type rhodopsin. Overall, our data support the role of the three cytoplasmic loops of rhodopsin and suggest that residues adjacent to the transmembrane helices are most important for transducin activation.     

High-resolution NMR structure of an AT-rich DNA sequence

We have determined, by proton NMR and complete relaxation matrix methods, the high-resolution structure of a DNA oligonucleotide in solution with nine contiguous AT base pairs. The stretch of AT pairs, TAATTATAATTATAATTA, is imbedded in a 27-nucleotide stem-and-loop construct, which is stabilized by terminal GC base pairs and an extraordinarily stable DNA loop GAA (Hirao et al., 1994, Nucleic Acids Res. 22, 576–582). The AT-rich sequence has three repeated TAATTA motifs, one in the reverse orientation. Comparison of the local conformations of the three motifs shows that the sequence context has a minor effect here: atomic RMSD between the three TAATTA fragments is 0.4–0.5 Å, while each fragment is defined within the RMSD of 0.3–0.4 Å. The AT-rich stem also contains a consensus sequence for the Pribnow box, TATAAT. The TpA, ApT, and TpTApA steps have characteristic local conformations, a combination of which determines a unique sequence-dependent pattern of minor groove width variation. All three TpA steps are locally bent in the direction compressing the major groove of DNA. These bends, however, compensate each other, because of their relative position in the sequence, so that the overall helical axis is essentially straight.     

Betam,a structural member of the X,K-ATPase beta subunit family,resides in the ER and does not associate with any known X,K-ATPase alpha subunit

betam, a muscle-specific protein, is structurally closely related to the X,K-ATPase beta subunits, but its intrinsic function is not known. In this study, we have expressed betam in Xenopus oocytes and have investigated its biosynthesis and processing as well as its putative role as a chaperone of X,K-ATPase alpha subunits, as a regulator of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), or as a Ca(2+)-sensing protein. Our results show that betam is stably expressed in the endoplasmic reticulum (ER) in its core glycosylated, partially trimmed form. Both full-length betam, initiated at Met(1), and short betam species, initiated at Met(89), are detected in in vitro translations as well as in Xenopus oocytes. betam cannot associate with and stabilize Na,K-ATPase (NK), or gastric and nongastric H,K-ATPase (HK) alpha isoforms. betam neither assembles stably with SERCA nor is its trypsin sensitivity or electrophoretic mobility influenced by Ca(2+). A mutant, in which the distinctive Glu-rich regions in the betam N-terminus are deleted, remains stably expressed in the ER and can associate with, but not stabilize X,K-ATPase alpha subunits. On the other hand, a chimera in which the ectodomain of betam is replaced with that of beta1 NK associates efficiently with alpha NK isoforms and produces functional Na,K-pumps at the plasma membrane. In conclusion, our results indicate that betam exhibits a cellular location and functional role clearly distinct from the typical X,K-ATPase beta subunits.     

Structure and properties of avian small heat shock protein with molecular weight 25 kDa

The primary structure of chicken small heat shock protein (sHsp) with apparent molecular weight 25 kDa was refined and it was shown that this protein has conservative primary structure 74RALSRQLSSG(83) at Ser77 and Ser81, which are potential sites of phosphorylation. Recombinant wild-type chicken Hsp25, its three mutants, 1D (S15D), 2D (S77D+S81D) and 3D (S15D+S77D+S81D), as well as delR mutant with the primary structure 74RALS-ELSSG(82) at potential sites of phosphorylation were expressed and purified. It has been shown that the avian tissues contain three forms of Hsp25 having pI values similar to that of the wild-type protein, 1D and 2D mutants that presumably correspond to nonphosphorylated, mono- and di-phosphorylated forms of Hsp25. Recombinant wild-type protein, its 1D mutant and Hsp25, isolated from chicken gizzard, form stable high molecular weight oligomeric complexes. The delR, 2D and 3D mutants tend to dissociate and exist in the form of a mixture of high and low molecular weight oligomers. Point mutations mimicking phoshorylation decrease chaperone activity of Hsp25 measured by reduction of dithiothreitol induced aggregation of alpha-lactalbumin, but increase the chaperone activity of Hsp25 measured by heat induced aggregation of alcohol dehydrogenase. It is concluded that avian Hsp25 has a more stable quaternary structure than its mammalian counterparts and mutations mimicking phosphorylation differently affect chaperone activity of avian Hsp25, depending on the nature of target protein and the way of denaturing.     

Twitchin, a thick-filament protein from molluscan catch muscle, interacts with F-actin in a phosphorylation-dependent way

Twitchin belongs to the titin-like giant proteins family, it is co-localized with thick filaments in molluscan catch muscles and regulates the catch state depending on its level of phosphorylation. The mechanism by which twitchin controls the catch state remains to be established. We report for the first time the ability of twitchin to interact with F-actin. The interaction is observed at low and physiological ionic strengths, irrespective of the presence or absence of Ca(2+). It was demonstrated by viscosity and turbidity measurements, low- and high-speed co-sedimentation, and with the light-scattering particle size analysis revealing the specific twitchin-actin particles. The twitchin-actin interaction is regulated by twitchin phosphorylation: in vitro phosphorylated twitchin does not interact with F-actin. We speculate that the catch muscle twitchin might provide a mechanical link between thin and thick filaments, which contributes to catch force maintenance.     

Microbial community structure in methane hydrate-bearing sediments of freshwater Lake Baikal

Gas hydrates in marine sediments have been known for many years but recently hydrates were found in the sediments of Lake Baikal, the largest freshwater basin in the world. Marine gas hydrates are associated with complex microbial communities involved in methanogenesis, methane oxidation, sulfate reduction and other biotransformations. However, the contribution of microorganisms to the formation of gas hydrates remains poorly understood. We examined the microbial communities in the hydrate-bearing sediments and water column of Lake Baikal using pyrosequencing of 16S rRNA genes. Aerobic methanotrophic bacteria dominated the water sample collected at the lake floor in the hydrate-bearing site. The shallow sediments were dominated by Archaea. Methanogens of the orders Methanomicrobiales and Methanosarcinales were abundant, whereas representatives of archaeal lineages known to perform anaerobic oxidation of methane, as well as sulfate-reducing bacteria, were not found. Affiliation of archaea to methanogenic rather than methane-oxidizing lineages was supported by analysis of the sequences of the methyl coenzyme M reductase gene. The deeper sediments located at 85-90 cm depth close to the hydrate were dominated by Bacteria, mostly assigned to Chloroflexi, candidate division JS1 and Caldiserica. Overall, our results are consistent with the biological origin of methane hydrates in Lake Baikal.     

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu⁵⁴³-Asp⁵⁴⁴ in this linker or Leu⁶¹¹, Arg⁶⁸³, or Phe⁶⁹⁴ in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.The Janus family of tyrosine kinases (JAKs)² are key regulators of cytokine receptor signaling in hematopoiesis and immune responses (1). Of the four mammalian JAK kinases, JAK2 transmits signals for a variety of cytokine receptors, including the erythropoietin receptor (EpoR) that is essential for red blood cell production (2). Upon Epo stimulation, JAK2 activates downstream signaling, such as STAT5, Ras/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/AKT pathways (2). Mice deficient in Epo, EpoR, or JAK2 die embryonically due to the absence of definitive erythropoiesis (3–5).In addition to regulation by phosphatases and suppressors of cytokine signaling (6, 7), JAK2 kinase activity is critically controlled by an autoinhibitory mechanism. Like other JAK members, JAK2 contains an N-terminal segment followed by a pseudokinase domain and a C-terminal tyrosine kinase domain. The N-terminal segment, consisting of a FERM (protein 4.1, ezrin, moezin, radixin homologous) domain and an atypical SH2 domain (1), mediates association with the membrane-proximal region of the cytokine receptors (8). Binding of JAK2 through its N-terminal segment to the EpoR is essential for EpoR surface expression (9). The pseudokinase domain is predicted to adopt a kinase fold but lacks residues essential for catalysis (10). Deletion of the pseudokinase domain leads to a marked increase in JAK2 kinase activity and loss of response to cytokine stimulation (11–13). Therefore, this domain is essential for JAK2 autoinhibition and is essential for JAK2 activation upon cytokine stimulation. Consistent with this notion, a point mutation in the JAK2 pseudokinase domain was identified in the majority of myeloproliferative disorder patients, including 90% of polycythemia vera (PV) patients (14–18). This mutation, V617F, in the presence of a dimerized receptor scaffold, such as the EpoR, resulted in the constitutive activation of JAK2 and downstream signaling effectors (19, 20) and caused erythrocytosis in a murine bone marrow transplant model (14, 21–23). Recently, mutations immediately adjacent to the JAK2 pseudokinase domain in the SH2-pseudokinase domain linker were identified in PV patients and shown to cause constitutive activation of JAK2 and a PV-like phenotype in mice (24–26). The molecular mechanisms underlying the control of JAK2 activity (i.e. the swift augmentation of its activity upon receptor activation) are poorly understood. The residues involved in the autoinhibition in JAK2 are unknown.In this work, we sought to characterize the regulatory mechanisms controlling JAK2 kinase activity. Using a functional screen for activating JAK2 mutations that signal constitutively, we discovered 13 mutations in the pseudokinase domain and in the SH2-pseudokinase domain linker. These mutations identified specific residues that are important for the inhibition of basal JAK2 kinase activity and for cytokine-induced JAK2 activation. In addition, we showed that the SH2-pseudokinase domain linker is essential for interaction with the EpoR, autoinhibitory regulation, and Epo-inducible JAK2 activation and may act as a switch in relaying cytokine receptor engagement to JAK2 activation by flexing the pseudokinase domain hinge.