Backbone and sidechain 1H, 15N and 13C assignments of Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa

The backbone and side chain resonance assignments of the Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa have been determined based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. This assignment is the first step towards the determination of the 3-dimensional structure of TpbA.     

1H, 13C, and 15N NMR assignments of an engineered intein based on Mycobacterium tuberculosis RecA

The backbone and side chain resonance assignments of an engineered intein based on Mycobacterium tuberculosis RecA have been determined based on triple-resonance experiments with the uniformly [¹³C,¹⁵N]-labeled protein.     

Synthesis of [19, 35, 36-13C3]-labeled TAK779 as a molecular probe

N,N-Dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride (TAK779) is a potent and selective non-peptide CCR5 antagonist. To use a site-specifically labeled form as a molecular probe, TAK779 containing ¹³C at positions C19, 35, and 36 was produced. A commercially available [¹³C]-methyl iodide was employed for the labeling. Starting from a known carboxylic acid segment containing no labeled carbon, the labeled TAK779 was constructed by the successive coupling of [¹³C]-labeled tolyl boronic ester by the Suzuki–Miyaura reaction and a [¹³C]-labeled aniline segment by amide bond formation.     

Backbone and sidechain 1H, 15N and 13C assignments of the NLRP7 pyrin domain

The resonance assignments of the human NLRP7 PYD domain have been determined based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. This assignment is the first step towards the 3D structure determination of the NLRP7 PYD domain.     

1H, 15N and 13C assignments of the calcium bound S100P

To determine the three-dimensional solution structure of the calcium bound S100P protein, the backbone and side chain resonance assignments of the S100P protein have been reported based on triple-resonance experiments using uniformly [¹³C, ¹⁵N]-labeled calcium bound protein.     

Backbone and sidechain 1H, 15N and 13C assignments of the human G-actin binding protein profilin IIa

The resonance assignment of the human profilin IIa have been determined, based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. These assignments facilitate further studies of interactions between profilin IIa and its poly-l-proline rich ligands.     

Backbone and side chain 1H, 15N and 13C assignments of the KSR1 CA1 domain

The backbone and side chain resonance assignments of the murine KSR1 CA1 domain have been determined based on triple-resonance experiments using uniformly [¹³C, ¹⁵N]-labeled protein. This assignment is the first step towards the determination of the three-dimensional structure of the unique KSR1 CA1 domain.     

NMR assignment of a KlbA intein precursor from Methanococcus jannaschii

The backbone and side chain resonance assignments of a precursor of the KlbA intein from Methanococcus jannaschii have been determined, based on triple-resonance experiments with the uniformly [¹³C,¹⁵N]-labeled protein.     

1H, 15N and 13C backbone resonance assignment of Rv1567c, an integral membrane protein from Mycobacterium tuberculosis

We report here the backbone assignment of Rv1567c, an integral membrane protein from Mycobacterium tuberculosis. The backbone resonance assignments were determined based on triple-resonance experiments with uniformly [¹³C,¹⁵N]-labeled protein in LMPG detergent micelles.     

Simultaneous acquisition of [13C,15N]- and [15N,15N]-separated 4D gradient-enhanced NOESY spectra in proteins

Summary The simultaneous acquisition of a 4D gradient-enhanced and sensitivity-enhanced [¹³C,¹⁵N]/[¹⁵N,¹⁵N]-separated NOESY is presented for the 74-residue [¹³C,¹⁵N]-labeled N-terminal SH3 domain of mGrb2 complexed with a peptide gragment from mSOS-2 in 90% H₂O. The method readily accommodates different ¹³C and ¹⁵N spectral widths, but requires that the same number of increments be collected for both ¹³C and ¹⁵N in the simultaneous dimension (F₂). For purposes of display and analysis, the two 4D spectra can be deconvolved during the processing stage by the appropriate linear combination of separately stored FIDs. Compared to collecting each of these two 4D data sets separately, the presented method is a factor (2)^1/2 more efficient in sensitivity per unit acquisition time. The interleaved nature of this method may also lead to improved peak registration between the two 4D spectra.     

Sequence-specific backbone 1H, 13C and 15N assignments of the 34 kDa catalytic domain of PTPN5 (STEP)

PTPN5 is a protein tyrosine phosphatase that plays an integral role in regulating excitatory postsynaptic activity. The sequence-specific backbone assignments of the murine PTPN5 catalytic domain have been determined based on triple-resonance experiments using uniformly [²H,¹³C,¹⁵N]-labeled protein.     

Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog (Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk⁺ hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk⁺ hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk⁺ hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C sequence specific backbone assignment of the vanadate inhibited hematopoietic tyrosine phosphatase

The sequence-specific backbone assignment of hematopoietic protein tyrosine phosphatase (HePTP; PTPN7) in presence of vanadate has been determined, based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. These assignments facilitate further studies of HePTP in the presence of inhibitors to target leukemia and provide further insights into the function of protein tyrosine phosphatases.     

NMR assignment of the nonstructural protein nsp3(1066–1181) from SARS-CoV

Sequence-specific NMR assignments of the globular core comprising the residues 1066–1181 within the non-structural protein nsp3e from the SARS coronavirus have been obtained using triple-resonance NMR experiments with the uniformly [¹³C, ¹⁵N]-labeled protein. The backbone and side chain assignments are nearly complete, providing the basis for the ongoing NMR structure determination. A preliminary identification of regular secondary structures has been derived from the ¹³C chemical shifts.     

Evolution of hedgehog and hedgehog-related genes, their origin from Hog proteins in ancestral eukaryotes and discovery of a novel Hint motif

Background

The Hedgehog (Hh) signaling pathway plays important roles in human and animal development as well as in carcinogenesis. Hh molecules have been found in both protostomes and deuterostomes, but curiously the nematode Caenorhabditis elegans lacks a bona-fide Hh. Instead a series of Hh-related proteins are found, which share the Hint/Hog domain with Hh, but have distinct N-termini.

Results

We performed extensive genome searches of the cnidarian Nematostella vectensis and several nematodes to gain further insights into Hh evolution. We found six genes in N. vectensis with a relationship to Hh: two Hh genes, one gene with a Hh N-terminal domain fused to a Willebrand factor type A domain (VWA), and three genes containing Hint/Hog domains with distinct novel N-termini. In the nematode Brugia malayi we find the same types of hh-related genes as in C. elegans. In the more distantly related Enoplea nematodes Xiphinema and Trichinella spiralis we find a bona-fide Hh. In addition, T. spiralis also has a quahog gene like C. elegans, and there are several additional hh-related genes, some of which have secreted N-terminal domains of only 15 to 25 residues. Examination of other Hh pathway components revealed that T. spiralis - like C. elegans - lacks some of these components. Extending our search to all eukaryotes, we recovered genes containing a Hog domain similar to Hh from many different groups of protists. In addition, we identified a novel Hint gene family present in many eukaryote groups that encodes a VWA domain fused to a distinct Hint domain we call Vint. Further members of a poorly characterized Hint family were also retrieved from bacteria.

Conclusion

In Cnidaria and nematodes the evolution of hh genes occurred in parallel to the evolution of other genes that contain a Hog domain but have different N-termini. The fact that Hog genes comprising a secreted N-terminus and a Hog domain are also found in many protists suggests that this gene family must have arisen in very early eukaryotic evolution, and eventually gave rise to hh and hh-related genes in animals. The results indicate a hitherto unsuspected ability of Hog domain encoding genes to evolve new N-termini. In one instance in Cnidaria, the Hh N-terminal signaling domain is associated with a VWA domain and lacks a Hog domain, suggesting a modular mode of evolution also for the N-terminal domain. The Hog domain proteins, the inteins and VWA-Vint proteins represent three different families of Hint domain proteins that evolved in parallel in eukaryotes.     

Complete 1H, 15N and 13C assignments, secondary structure, and topology of recombinant human interleukin-6

Summary Essentially complete backbone and side-chain ¹H, ¹⁵N and ¹³C resonance assignments for the 185-aminoacid cytokine interleukin-6 (IL-6) are presented. NMR experiments were performed on uniformly [¹⁵N]-and [¹⁵N, ¹³C]-labeled recombinant human IL-6 (rIL-6) using a variety of heteronuclear NMR experiments. A combination of ¹³C-chemical shift, amide hydrogen-bond exchange, and ¹⁵N-edited NOESY data allowed for analysis of the secondary structure of IL-6. The observed secondary structure of IL-6 is composed of loop regions connecting five -helices, four of which are consistent in their length and disposition with the four-helix bundle motif present in other related cytokines and previously postulated for IL-6. In addition, the topology of the overall fold was found to be consistent with a left-handed up-up-down-down four-helix bundle based on a number of long-range interhelical NOEs. The results presented here provide deeper insight into structure-function relationships among members of the four-helix bundle family of proteins.     

Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL⁺ CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL⁺ CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL⁺ cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL⁺ cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved ¹³C and ¹⁵N signals of [1-¹³C]Tyr-, [¹⁵N]Pro- and [1-¹³C]Val-, [¹⁵N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-¹³C]Val-, [¹⁵N]Pro signals to the specific residues in bR. The previous assignments of [1-¹³C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent ¹⁵N chemical shifts of Pro bonded to Val in the presence of ¹³C-¹⁵N correlation, although no assignment of peak is feasible for ¹³C nuclei not bonded to Pro. ¹³C or ¹⁵N spin-lattice relaxation times (T₁), spin-spin relaxation times under the condition of CP-MAS (T₂), and cross relaxation times (T_CH and T_NH) for ¹³C and ¹⁵N nuclei and carbon or nitrogen-resolved, ¹H spin-lattice relaxation times in the rotating flame (¹H T_1ρ) for the assigned signals were measured in [1-¹³C]Val-, [¹⁵N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid β-sheet in spite of longer loop and possesses large amplitude motions as revealed from ¹³C and ¹⁵N conformation-dependent chemical shifts and T₁, T₂, ¹H T_1ρ and cross relaxation times. In addition, breakage of the β-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

Distinct Effects of the mesenchymal dysplasia Gene Variant of Murine Patched-1 Protein on Canonical and Non-canonical Hedgehog Signaling Pathways

Hedgehog (Hh) signaling requires regulation of the receptor Patched-1 (Ptch1), which, in turn, regulates Smoothened activity (canonical Hh signaling) as well as other non-canonical signaling pathways. The mutant Ptch1 allele mesenchymal dysplasia (mes), which truncates the Ptch1 C terminus, produces a limited spectrum of developmental defects in mice as well as deregulation of canonical Hh signaling in some, but not all, affected tissues. Paradoxically, mes suppresses canonical Hh signaling and binds to Hh ligands with an affinity similar to wild-type mouse Ptch1 (mPtch1). We characterized the distinct activities of the mes variant of mPtch1 mediating Hh signaling through both canonical and non-canonical pathways. We demonstrated that mPtch1 bound c-src in an Hh-regulated manner. Stimulation with Sonic Hedgehog (Shh) of primary mammary mesenchymal cells from wild-type and mes animals activated Erk1/2. Although Shh activated c-src in wild-type cells, c-src was constitutively activated in mes mesenchymal cells. Transient assays showed that wild-type mPtch1, mes, or mPtch1 lacking the C terminus repressed Hh signaling in Ptch1-deficient mouse embryo fibroblasts and that repression was reversed by Shh, revealing that the C terminus was dispensable for mPtch1-dependent regulation of canonical Hh signaling. In contrast to these transient assays, constitutively high levels of mGli1 but not mPtch1 were present in primary mammary mesenchymal cells from mes mice, whereas the expression of mPtch1 was similarly induced in both mes and wild-type cells. These data define a novel signal transduction pathway involving c-src that is activated by the Hh ligands and reveals the requirement for the C terminus of Ptch in regulation of canonical and non-canonical Hh signaling pathways.