Insights into signal transduction revealed by the low resolution structure of the FixJ response regulator

P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase inhibitors of CDK4 and CDK6, which play a key role in human cell cycle control. P19 comprises ten alpha-helices arranged sequentially in five ankyrin repeats forming an elongated structure. This rather simple topology, combined with its physiological function, makes p19 an interesting model protein for folding studies. Urea-induced unfolding transitions monitored by far-UV CD and phenylalanine fluorescence coincide and suggest a two-state mechanism for equilibrium unfolding. Unfolding of p19 followed by 2D (1)H-(15)N HSQC spectra revealed a third species at moderate urea concentrations with a maximum population of about 30 % near 3.2 M urea. It shows poor chemical shift dispersion, but cross-peaks emerge for some residues that are distinct from the native or unfolded state. This equilibrium intermediate either arises only at high protein concentrations (as in the NMR experiment) or has similar optical properties to the unfolded state. Stopped-flow far-UV CD experiments at various urea concentrations revealed that alpha-helical structure is formed in three phases, of which only the fastest phase (10 s(-1)) depends upon the urea concentration. The kinetic of the slowest phase (0.017 s(-1)) can be resolved by 1D real-time NMR and accelerated by cyclophilin. It is limited in rate by prolyl isomerization, and native-like ordered structure cannot form prior to this isomerization. The two fast phases lead to 83 % native protein within the dead time of the NMR experiment. In contrast to p16(INK4a), which exhibits only a marginal stability and high unfolding rates, p19 shows the expected stability for a protein of this size with a clear kinetic barrier between the unfolded and folded state. Therefore, p19 might complement the function of less stable INK4 inhibitors in cell cycle control under unfavorable conditions.     

Solution structure of a llama single-domain antibody with hydrophobic residues typical of the VH/VL interface

The three-dimensional structure of a llama single-domain antibody BrucD4-4 was established by use of solution NMR spectroscopy. BrucD4-4 has Val, Gly, Leu, and Trp residues at positions 37, 44, 45, and 47, which are considered to be a hallmark to distinguish llama VH from V(H)H fragments at the germline level. In contrast to the murine and human VHs, BrucD4-4 has sufficient solubility, is monomeric in solution, and displays high-quality NMR spectra characteristic of well-structured proteins. Amide proton/deuterium exchange and the (15)N relaxation data showed that BrucD4-4 has a classic protein structure with a well-packed core and comparatively mobile surface loops. The three-dimensional architecture of BrucD4-4 is analogous to that of VHs from murine and human F(v)s and camelid V(H)Hs with two pleated beta-sheets formed by four and five beta-strands. A canonical and undistorted beta-barrel exposes a number of hydrophobic residues into the solvent on the surface of the three-dimensional structure. The eight-residue H3 loop folds over the side chain of Val37 similarly to that in llama V(H)Hs; however, this interaction may be transient due to the H3 conformational flexibility. Overall, the surface characteristics of BrucD4-4 with respect to hydrophobicity appear to lie between the human VH domain from Fv Pot and the llama V(H)H fragment HC-V, which may explain its enhanced solubility allowing NMR structural analysis.     

Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases

Three key regulatory enzymes in ganglioside biosynthesis, sialyltransferase I (ST1), sialyltransferase II (ST2), and N-acetylgalactosaminyltransferase I (GalNAcT), have been expressed as fusion proteins with green, yellow, or red fluorescent protein (GFP, YFP, or RFP) in F-11A cells. F-11A cells are a substrain of murine neuroblastoma F-11 cells that contain only low endogenous ST2 and GalNAcT activity. The subcellular localization of the fusion proteins has been determined by fluorescence microscopy, and the ganglioside composition of these cells was analyzed by high-performance thin-layer chromatography (HPTLC). ST2-GFP (85 kDa) shows a distinct Golgi localization, whereas ST1-YFP (85 kDa) and GalNAcT-RFP (115 kDa) are broadly distributed in ER and Golgi. Untransfected F-11A cells contain mainly GM3, whereas stable transfection with ST2 or GalNAcT results in the predominant expression of b-series complex gangliosides (BCGs). This result indicates that the expression of ST2 enhances the activity of endogenous GalNAcT and vice versa. The specificity of this reaction has been verified by in vitro activity assays with detergent-solubilized enzymes, suggesting the formation of an enzyme complex between ST2 and GalNAcT but not with ST1. Complex formation has also been verified by co-immunoprecipitation of ST2-GFP upon transient transfection with GalNAcT-HA-RFP and by GFP-to-RFP FRET signals that are confined to the Golgi. FRET analysis also suggests that ST2-GFP binds tightly to pyrene-labeled GM3 but not to ST1. We hypothesize that an ST2-GM3 complex is associated with GalNAcT, resulting in the enhanced conversion of GM3 to GD3 and BCGs in the Golgi. Taken together, our results support the concept that ganglioside biosynthesis is tightly regulated by the formation of glycosyltransferase complexes in the ER and/or Golgi.     

Evolutionary History of 4.5SI RNA and Indication That It Is Functional

To date, the small nuclear 4.5S_I RNA has only been studied in the rat (Rattus norvegicus). Combining PCR and hybridization analyses, we have revealed 4.5S_I RNA homologues sequences in the genomes of four myomorph rodent families (Muridae, Cricetidae, Spalicidae, and Rhizomyidae), and not in other myomorph families (Dipodidae, Zapodidae, Geomyidae, and Heteromyidae) or sciuromorph and caviomorph rodents. By Northern-hybridization, 4.5S_I RNA has been detected in the common rat (R. norvegicus, Muridae), golden hamster (Mesocricetus auratus, Cricetidae), and Russian mole rat (Spalax microphthalmus, Spalacidae), but not in the related great jerboa (Allactaga jaculus, Dipodidae) or in four non-myomorph rodent species tested. cDNA derived from 4.5S_I RNA of M. auratus and S. microphthalmus has been cloned and sequenced. The hamster RNA is found to differ from rat 4.5S_I RNA by only one nucleotide substitution. For the mole rat, two variants of 4.5S_I RNA are detected: short (S) and long (L) with length 101 and 108 nt, respectively. The L variant differs from the S variant as well as from murid and cricetid 4.5S_I RNAs by both a 7 nt insertion and a varying number of nucleotide substitutions. The sequence similarity between the spalacid S-variant and murid/crecitid variants of 4.5S_I RNA is 90%. Judging from species distribution, 4.5S_I RNA genes emerged during the same period of time as the related short interspersed element B2 arose. This occurred after the divergence of Dipodidae lineage but before the branching of Spalicidae/Rhizomyidae lineage from a common myomorph rodent stem. S variant genes seemed to emerge in a common ancestor of spalacids and rhizomyds whereas L variant genes formed in spalacids following the divergence of these two families. The low rate of evolutionary changes of 4.5S_I RNA, at least, in murids and cricetids (6 × 10⁻⁴ substitutions per site per million years), suggests that this RNA is under selection constraint and have a function. This is a remarkable fact if the recent origin and narrow species distribution range of 4.5S_I RNA genes is taken into account. Genes with narrow species distribution are proposed to be referred to as stenogenes. Received: 11 December 2000 / Accepted: 27 August 2001     

Wiring optimization in cortical circuits

Wiring a brain presents a formidable problem because neural circuits require an enormous number of fast and durable connections. We propose that evolution was likely to have optimized neural circuits to minimize conduction delays in axons, passive cable attenuation in dendrites, and the length of "wire" used to construct circuits, and to have maximized the density of synapses. Here we ask the question: "What fraction of the volume should be taken up by axons and dendrites (i.e., wire) when these variables are at their optimal values?" The biophysical properties of axons and dendrites dictate that wire should occupy 3/5 of the volume in an optimally wired gray matter. We have measured the fraction of the volume occupied by each cellular component and find that the volume of wire is close to the predicted optimal value.     

GABAA receptors at hippocampal mossy fibers

Presynaptic GABAA receptors modulate synaptic transmission in several areas of the CNS but are not known to have this action in the cerebral cortex. We report that GABAA receptor activation reduces hippocampal mossy fibers excitability but has the opposite effect when intracellular Cl- is experimentally elevated. Synaptically released GABA mimics the effect of exogenous agonists. GABAA receptors modulating axonal excitability are tonically active in the absence of evoked GABA release or exogenous agonist application. Presynaptic action potential-dependent Ca2+ transients in individual mossy fiber varicosities exhibit a biphasic dependence on membrane potential and are altered by GABAA receptors. Antibodies against the alpha2 subunit of GABAA receptors stain mossy fibers. Axonal GABAA receptors thus play a potentially important role in tonic and activity-dependent heterosynaptic modulation of information flow to the hippocampus.     

Na2CO3 influence on DNA double helix stability: strong anion destabilizing effect

Addition of Na(2)CO(3) to almost salt-free DNA solution (5.10(-5)M EDTA, pH=5.7, T(m)=26.5 degrees C) elevates both pH and the DNA melting temperature (T(m)) if Na(2)CO(3) concentration is less than 0.004 M. For 0.004 M Na(2)CO(3), T(m)=58 degrees C is maximal and pH=10.56. Further increase in concentration gives rise to a monotonous decrease in T(m) to 37 degrees C for 1M Na(2)CO(3) (pH=10.57). Increase in pH is also not monotonous. The highest pH=10.87 is reached at 0.04 M Na(2)CO(3) (T(m)=48.3 degrees C). To reveal the cause of this DNA destabilization, which happens in a narrow pH interval (10.56/10.87) and a wide Na(2)CO(3) concentration interval (0.004/1M), a procedure has been developed for determining the separate influences on T(m) of Na(+), pH, and anions formed by Na(2)CO(3) (HCO(3)(-) and CO(3)(2-)). Comparison of influence of anions formed by Na(2)CO(3) on DNA stability with Cl(-) (anion inert to DNA stability), ClO(4)(-) (strong DNA destabilizing "chaotropic" anion) and OH(-) has been carried out. It has been shown that only Na(+) and pH influence T(m) in Na(2)CO(3) solution at concentrations lower than 0.001 M. However, the T(m) decrease with concentration for [Na(2)CO(3)]>/=0.004 M is only partly caused by high pH=10.7. Na(2)CO(3) anions also exert a strong destabilizing influence at these concentrations. For 0.1M Na(2)CO(3) (pH=10.84, [Na(+)]=0.2M, T(m)=42.7 degrees C), the anion destabilizing effect is higher 20 degrees C. For NaClO(4) (ClO(4)(-) is a strong "chaotropic" anion), an equal anion effect occurs at much higher concentrations approximately 3M. This means that Na(2)CO(3) gives rise to a much stronger anion effect than other salts. The effect is pH dependent. It decreases fivefold at neutral pH after addition of HCl to 0.1M Na(2)CO(3) as well as after addition of NaOH for pH greater than 11.2.