Proteomic identification of brain proteins that interact with dynein light chain LC8

Cytoplasmic dynein is a large minus end-directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two-hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain-associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post-synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.     

Thermal degradation of allicin in garlic extracts and its implication on the inhibition of the in-vitro growth of Helicobacter pylori

Allicin, the main active principle related to Allium sativum chemistry, is considered to be responsible for the bacteriostatic properties of garlic. The work described here has demonstrated the direct implication of the allicin present in solvent-free garlic extracts obtained with ethanol (ethanolic garlic extract, EGE) and acetone (acetonic garlic extract, AGE) in the inhibition of the in-vitro growth of Helicobacter pylori (Hp), the bacterium responsible for serious gastric diseases such as ulcers and even gastric cancer. The evolution of allicin concentration as a function of time and temperature has been the subject of a kinetic study. The reaction order, activation energy, and preexponential factor (in accordance with Arrhenius theory) have been determined for the decomposition process of allicin in these organic media. First-order decomposition, an activation energy of 97.4 kJ/mol, and an Arrhenius preexponential factor of 8.9 x 10(10) s(-1) have been determined for allicin in EGE. For allicin in AGE the kinetic order determined was 1.5, the activation energy 184.5 kJ/mol, and the preexponential factor 3.1 x 10(24) s(-1) (mg/L)(-0.5). The presence or absence of allicin in these garlic products was found to be crucial for the inhibition of the in-vitro growth of Hp, as demonstrated by microbiological analysis for AGE. A relationship has been identified between the effectiveness and durability of the anti-Hp properties shown by AGE and the allicin content of these products. The bacteriostatic properties were active for up to 10 months if the samples were maintained at 6 degrees C.     

Benzimidazole derivatives. Part 5: design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT1A/5-HT3 ligands

A series of new mixed benzimidazole-arylpiperazine derivatives were designed by incorporating in general structure III the pharmacophoric elements of 5-HT(1A) and 5-HT(3) receptors. Compounds 1-11 were synthesized and evaluated for binding affinity at both serotoninergic receptors, all of them exhibiting high 5-HT(3)R affinity (K(i)=10-62nM), and derivatives with an o-alkoxy group in the arylpiperazine ring showing nanomolar affinity for the 5-HT(1A)R (K(i)=18-150nM). Additionally, all the synthesized compounds were selective over alpha(1)-adrenergic and dopamine D(2) receptors (K(i)>1000-10,000nM). Compound 3 was selected for further pharmacological characterization due to its interesting binding profile as mixed 5-HT(1A)/5-HT(3) ligand with high affinity for both receptors (5-HT(1A): K(i)=18.0nM, 5-HT(3): K(i)=27.2nM). In vitro and in vivo findings suggest that this compound acts as a partial agonist at 5-HT(1A)Rs and as a 5-HT(3)R antagonist. This novel mixed 5-HT(1A)/5-HT(3) ligand was also effective in preventing the cognitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test, suggesting a potential interest in the treatment of cognitive dysfunction.     

Interleukin-1 beta (IL-1beta) induces tumor necrosis factor alpha (TNF-alpha) expression on mouse myeloid multipotent cell line 32D cl3 and inhibits their proliferation

Interleukin-1 alpha (IL-1alpha) and beta (IL-1beta) are well known factors that stimulate hematopoiesis, nevertheless there are reports that show that they can also inhibit this activity. While both IL-1alpha and IL-1beta induce the expression of hematopoietic cytokines, such as growth factors and their receptors on myeloid cells, helping thus to regulate hematopoiesis, it is not known if their inhibitory activity is also mediated through the induction of other specific cytokines. In this work we show that recombinant human IL-1beta (rhIL-1beta) inhibits the proliferation of a mouse IL-3-dependent myeloid multipotent cell line (32D cl3), without inducing its differentiation. We show that rhIL-1beta induces in 32D cl3 cells the expression of the tumor necrosis factor alpha (TNF-alpha) gene, a well known growth inhibitor, and that the rhIL-1beta growth inhibition property on 32D cl3 cells is partially due to this secreted TNF-alpha, hinting thus that the inhibition of hematopoiesis by IL-1 is mediated through other induced cytokines.     

In vitro effects of LPS, IL-2, PDGF and CRF on haemocytes of Mytilus galloprovincialis Lmk

The cells in charge of the innate immune response in the marine mussel Mytilus galloprovincialis Lmk. are the haemocytes. These cells respond in different ways to agents such as lipopolysaccharide (LPS), interleukin-2 (IL-2), platelet-derived growth factor (PDGF) and corticotropin releasing factor (CRF). After stimulation of the haemocytes, the expression of molecules reactive with monoclonal antibodies raised to the alpha chain of the IL-2 receptor, present in their membrane, differed depending on the agent used. The same happened with regard to the levels of dopamine, adrenaline and noradrenaline released to the medium by the haemocytes. It should also be noted that no catecholamine release was detected and the level of expression of IL-2Ralpha showed no significant variation in cultured cells that had not been treated with inducers. These facts would indicate that most haemocytes were in the same starting condition at the moment that the stimulation was performed. Therefore, cultured haemocytes can be a highly reliable model in the study of the innate immune system.     

A novel locus for autosomal dominant nonsyndromic hearing loss (DFNA44) maps to chromosome 3q28–29

Hereditary non-syndromic sensorineural hearing loss (NSSHL) is a genetically highly heterogeneous group of disorders. Autosomal dominant forms account for up to 20% of cases. To date, 39 loci have been identified by linkage analysis of affected families that segregate NSSHL forms in the autosomal dominant mode (DFNA). Investigation of a large Spanish pedigree with autosomal dominant inheritance of bilateral and progressive NSSHL of postlingual onset excluded linkage to known DFNA loci and, in a subsequent genome-wide scan, the disorder locus was mapped to 3q28-29. A maximum two-point LOD score of 4.36 at theta=0 was obtained for marker D3S1601. Haplotype analysis placed the novel locus, DFNA44, within a 3-cM genetic interval defined by markers D3S1314 and D3S2418. Heteroduplex analysis and DNA sequencing of coding regions and exon/intron boundaries of two genes (CLDN16 and FGF12) in this interval did not reveal disease-causing mutations.     

Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8)

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.     

Evidence for sequential barriers and obligatory intermediates in apparent two-state protein folding

Many small proteins fold fast and without detectable intermediates. This is frequently taken as evidence against the importance of partially folded states, which often transiently accumulate during folding of larger proteins. To get insight into the properties of free energy barriers in protein folding we analyzed experimental data from 23 proteins that were reported to show non-linear activation free-energy relationships. These non-linearities are generally interpreted in terms of broad transition barrier regions with a large number of energetically similar states. Our results argue against the presence of a single broad barrier region. They rather indicate that the non-linearities are caused by sequential folding pathways with consecutive distinct barriers and a few obligatory high-energy intermediates. In contrast to a broad barrier model the sequential model gives a consistent picture of the folding barriers for different variants of the same protein and when folding of a single protein is analyzed under different solvent conditions. The sequential model is also able to explain changes from linear to non-linear free energy relationships and from apparent two-state folding to folding through populated intermediates upon single point mutations or changes in the experimental conditions. These results suggest that the apparent discrepancy between two-state and multi-state folding originates in the relative stability of the intermediates, which argues for the importance of partially folded states in protein folding.     

Catalase-peroxidase KatG of Burkholderia pseudomallei at 1.7A resolution

The catalase-peroxidase encoded by katG of Burkholderia pseudomallei (BpKatG) is 65% identical with KatG of Mycobacterium tuberculosis, the enzyme responsible for the activation of isoniazid as an antibiotic. The structure of a complex of BpKatG with an unidentified ligand, has been solved and refined at 1.7A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are 15.3% and 18.6%, respectively. The crystallized enzyme is a dimer with one modified heme group and one metal ion, likely sodium, per subunit. The modification on the heme group involves the covalent addition of two or three atoms, likely a perhydroxy group, to the secondary carbon atom of the vinyl group on ring I. The added group can form hydrogen bonds with two water molecules that are also in contact with the active-site residues Trp111 and His112, suggesting that the modification may have a catalytic role. The heme modification is in close proximity to an unusual covalent adduct among the side-chains of Trp111, Tyr238 and Met264. In addition, Trp111 appears to be oxidized on C(delta1) of the indole ring. The main channel, providing access of substrate hydrogen peroxide to the heme, contains a region of unassigned electron density consistent with the binding of a pyridine nucleotide-like molecule. An interior cavity, containing the sodium ion and an additional region of unassigned density, is evident adjacent to the adduct and is accessible to the outside through a second funnel-shaped channel. A large cleft in the side of the subunit is evident and may be a potential substrate-binding site with a clear pathway for electron transfer to the active-site heme group through the adduct.