Establishment of a cell model of ALS disease: Golgi apparatus disruption occurs independently from apoptosis.

The Golgi apparatus (GA) appears disrupted in motor neurons of amyotrophic lateral sclerosis (ALS). Here, mouse motor neuron-like NSC-34 cell lines stably expressing human superoxide dismutase 1 (hSOD1)(wt) and mutant hSOD1(G93A), as an ALS cell model, were constructed. The number of cells with disrupted GA increased from 14% to 34%. Furthermore, NSC-34/hSOD1(G93A) cells showed lower levels of proliferation and differentiation. GA disruption was not caused by apoptosis as determined by several techniques including caspase-3 activation. Similarly, spinal cords from ALS patients did not show caspase-3 activation. Therefore, NSC-34/hSOD1(G93A) cells are a suitable cell model to study GA dysfunction in ALS.

The [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough is a bacterial lipoprotein lacking a typical lipoprotein signal peptide.

Desulfovibrio vulgaris Hildenborough has a membrane-bound [NiFeSe] hydrogenase whose mode of membrane association was unknown since it is constituted by two hydrophilic subunits. This work shows that this hydrogenase is a bacterial lipoprotein bound to the membrane by lipidic groups found at the N-terminus of the large subunit, which is unusual since it is missing the typical lipoprotein signal peptide. Nevertheless, the large subunit has a conserved four residue lipobox and its synthesis is sensitive to the signal peptidase II inhibitor globomycin. The D. vulgaris [NiFeSe] hydrogenase is the first example of a bacterial lipoprotein translocated through the Tat pathway.

Production and purification of functional truncated soluble forms of human recombinant L1 cell adhesion glycoprotein from Spodoptera frugiperda Sf9 cells.

L1 is a human cell adhesion glycoprotein involved in the development of the central nervous system that comprises six immunoglobulin-like domains (Ig1-Ig6), five fibronectin-type III (FN1-FN5) domains, a single transmembrane region and a cytoplasmic domain. It contains 20 potential N-glycosylation sites and is heavily glycosylated in a variety of cell types. In this work, seven truncated soluble forms including L1 ectodomain (L1/ECD) and Ig domains 5-6 (L1/Ig5-6) have been constructed by PCR and have been cloned, as well as the full-length form (L1), in the stable expression vector for insect cells pMIB/V5-His-TOPO. Spodoptera frugiperda Sf9 cell lines expressing the truncated forms have been obtained, and all proteins were successfully secreted. L1/ECD and L1/Ig5-6 were produced in shake flasks with productions of 3 and 32 mg/L on the third and fourth day of culture, respectively. When L1/Ig5-6 was produced for four days in 2L bioreactor 200 mg/L protein were recovered from the supernatants on the fourth day of culture. Affinity-purified L1/ECD and L1/Ig5-6 were immobilized on poly-d-lysine coated coverslips, and were shown to be active in inducing neurite outgrowth from human NT2N neurons. Therefore, correctly folded and functional truncated forms of human L1 have been produced in high amounts from insect cells using a stable expression system.

Natural domain design: enhanced thermal stability of a zinc-lacking ferredoxin isoform shows that a hydrophobic core efficiently replaces the structural metal site.

Zinc centers play a key role as important structure determinants in a variety of proteins including ferredoxins (Fd). Here, we exploit the availability of two highly similar ferredoxin isoforms from the thermophile Sulfolobus metallicus, which differ in the residues involved in coordinating a His/Asp zinc site that ties together the protein core with its N-terminal extension, to investigate the effect of the absence of this site on ferredoxin folding. The conformational properties of the zinc-containing (FdA) and zinc-lacking (FdB) isoforms were investigated using visible absorption and tryptophan fluorescence emission. Fluorescence quenching studies, together with comparative modeling and molecular dynamics simulations, indicate that the FdB N-terminal extension assumes a fold identical to that of the Zn(2+)-containing isoform. The thermal stability of the isoforms was investigated in a broad pH range (2 < pH < 10), and at physiological pH conditions, both proteins unfold above 100 degrees C. Surprisingly, the Zn(2+)-lacking isoform was always found to be more stable than its Zn(2+)-containing counterpart: a DeltaT(m) approximately 9 degrees C is determined at pH 7, a difference that becomes even more significant at extreme pH values, reaching a DeltaT(m) approximately 24 degrees C at pH 2 and 10. The contribution of the Zn(2+) site to ferredoxin stability was further resolved using selective metal chelators. During thermal unfolding, the zinc scavenger TPEN significantly lowers the T(m) in FdA ( approximately 10 degrees C), whereas it has no effect in FdB. This shows that the Zn(2+) site contributes to ferredoxin stability but that FdB has devised a structural strategy that accounts for an enhanced stability without using a metal cross-linker. An analysis of the FdB sequence and structural model leads us to propose that the higher stability of the zinc-containing ferredoxin results from van der Waals contacts formed between the residues that occupy the same spatial region where the zinc ligands are found in FdA. These favor the formation of a novel local stabilizing hydrophobic core and illustrate a strategy of natural fold design.

Biochemical characterization of plasma in amyotrophic lateral sclerosis: amino acid and protein composition.

In this work, we have studied the amino acid and protein composition of the plasma from a group of 32 ALS patients. As controls, groups of 10 healthy subjects (HC) and 32 patients with other neuromuscular disorders have been analysed. When the HC group was compared with the ALS group there were significant decreases of His (39+/-18 to 24+/-9 microM, p<0.01) and Ala (313+/-62 to 237+/-66 microM, p<0.05), and a significant increase of Asn (89+/-41 to 118+/-24 microM, p<0.05), for the ALS group. When the three groups were compared, we observed significant decreased concentrations of Ser, His, Thr, Ala, Arg, Tyr, Met, Cys, Ile, and significant increases of Asn, Phe and Lys. An increase of proteolytic products of alpha2-macroglobulin (alpha2-M), an acute-phase serum glycoprotein that functions as a protease inhibitor, has been observed for a subgroup of ALS patients by Western blot. Furthermore, the detection of alpha2-M during disease progression has shown increases of the intact subunit and of a proteolytic product for two of the four patients analysed. Another acute-phase glycoprotein, haptoglobin, which regulates haemoglobin degradation, was not increased for the same group of patients. The results obtained suggested that diet supplementation with His and Ala and modulation of alpha2-M might have some beneficial effects on the course of ALS.

Characterization of a highly thermostable extracellular lipase from Lactobacillus plantarum.

After screening for the presence of lipase activity in lactobacilli isolated from "chouriço", a traditional Portuguese dry fermented sausage, a strain of Lactobacillus plantarum (DSMZ 12028) was chosen for extracellular lipase characterisation and purification. Proteinase K did not significantly affect lipolytic activity, as opposed to trypsin, which completely eliminated this activity. Among NaCl, Ca2+, EDTA, BSA, glycerol, Mn2+ and Mg2+, only Mn2+ and Mg2+ stimulated the lipase. Purification by gel filtration chromatography and gel electrophoresis revealed four bands, between 98 and 45 kDa, all with lipolytic activity against olive oil.