Can Nep1-like proteins form oligomers?

Nep1-like proteins (NLPs) are a novel family of microbial elicitors of plant necrosis that induce a hypersensitive-like response in dicot plants. The spatial structure and role of these proteins are yet unknown. In a paper published in BMC Plant Biology (2008; 8:50) we have proposed that the core region of Nep1-like proteins (NLPs) belong to the Cupin superfamily. Based on what is known about the Cupin superfamily, in this addendum to the paper we discuss how NLPs could form oligomers.Key words: quaternary structure, necrosis and ethylene inducing proteins, NLPs, MpNEP1, MpNEP2, NPP1, Moniliophthora perniciosa, Phytophthora parasiticaCupins may be organized as monomers, dimers, hexamers and octamers of β-barrel domains.¹ To the best of our knowledge trimers have not been detected yet. The interaction of two monomers building up a dimeric structure is basically performed by three types of interactions: hydrophobic interactions between β-strands in different subunits, salt bridges and hydrogen bonds between β-strands. In cupin dimers, the hydrophobic interactions occur between two βI strands in different subunits (Fig. 1A and B). This strand represents the central axis of rotation of the dimer as one residue in βI interacts with the corresponding residue in the other subunit (Fig. 1B). Therefore, all residues in βI must be hydrophobic, as one residue interacts with the other subunit and the next one in the sequence interacts with the interior of the protein. Charged residues in βI would disrupt such interactions. Most cupin dimers have strong hydrophobic residues such as tryptophan (W), phenylalanine (F) and methionine (M) pointing towards the own subunit (↓), while small hydrophobic residues such as leucine (L), isoleucine (I), and valine (V) point to the other subunit (↑). A particular case is leucine that interacts with other subunits, for instance, βI = liaW (positions 217–220 in Fig. 1B) and βI = LVsw of type I and II NLP consensuses, respectively. Therefore, the pattern of hydropathicity suggests that the side chain orientation is βI = l217 ↑ i218 ↓ a219 ↑ W220 ↓ d221 ↑. However we observe that just after βI there is a charged residue (aspartate D221) which would point outwards disrupting the dimer or at least making it less stable. It is interesting to observe that the requirement for a negatively charged residue at this last position is very high: 96% of all type I NLPs contains an aspartate (D) or glutamate (E) indicating an important role for it, maybe in avoiding dimerization of the NLPs. A second interesting hypothesis is as follows: several cupins are oxygenases, decarboxylases, etc. and use a negatively charged residue, such as aspartate or glutamate as proton donor.¹ Now, if the alternate pattern of side chains of the residues is βI = l217 ↓ i218 ↑ a219 ↓ W220 ↑ d221 ↓, instead of the previous one, then the aspartate or glutamate residue would point to the hydrophobic pocket and would be positioned to interact with the metal ion, as in cupins with enzymatic activity. However, there are no experimental evidences that the NLPs have enzymatic activity.Open in a separate windowFigure 1(A) Three-dimensional structure prediction for type I NLP consensus, (B) Interface between two βI strands in type I NLP consensus. From the left to the right: EF-coil with the conserved residue H162, βC and βH strands (superposed) with the conserved histidines H133 and H135 in βC, H193 and leucine L195 in βH, W220 in βI and W118 in βB. The strands in the right subunit follow the same pattern but rotated.The second type of interaction is salt bridges between charged residues in different subunits. Analyzing all interacting side chains in the 1VJ2 protein (dimer), we verify that the charged side chains of N35 and E57 (numbers in original structure) are only 2.72 Å apart. In the NLPs, this corresponds to N108^36% (Q108^60%) at the border of βB and E138^98%. The negatively charged residue D125 helps to correct the orientation of the subunits in relation to each other avoiding any disorientation. The high conservation level of these residues suggests that NLPs are dimeric structures. However, as we will see next, only hydrophobic and charged interactions are not enough to build a dimer.Garcia et al. (2007)² have used small angle X-ray scattering (SAXS) to show that, in solution, at low concentrations (<2 mg/ml) the two copies of the NLPs of Moniliophthora perniciosa, MpNEP1 and MpNEP2, exist as dimers and monomers, respectively. The same technique showed that at higher concentrations, >5 mg/ml, both proteins exist as dimers, as is the case for PpNPP1.² They also reported, based on electrophoresis analysis, that PpNPP1 and MpNEP1 exist as oligomers and MpNEP2 as monomers.² However, experiments with the PpNPP1 in size exclusion chromatography using myoglobin as size standard suggest that PpNPP1 is a monomer.³ Figure 2 compares MpNEP1, MpNEP2 and PpNPP1, where the most relevant differences in sequence are marked with asterisks (*) and are possibly related to the differences in oligomeric properties between MpNEP1 and PpNPP1 with MpNEP2. These positions are methionine M27 and leucine L35, which occur only in MpNEP2, glycine G250, which occurs only in MpNEP2 and NEP1 (Fusarium oxysporum) and lysine K31, which occurs only MpNEP2, {"type":"entrez-protein","attrs":{"text":"BAB04114","term_id":"10173008"}}BAB04114 (Bacillus halodurans) and {"type":"entrez-protein","attrs":{"text":"AAU23136","term_id":"52003194"}}AAU23136 (Bacillus licheniformis). The other residues are aspartate D28, which occurs 9 times and alanine A37 which occurs 7 times of all investigated NLPs. Thus, the sequence mdHDkiakl at the start of the NLPs seems to explain the monomeric state of MpNEP2, although at higher concentrations they form dimers. Besides the weak hydrophobic interactions, dimeric cupins and bicupins (two β barrels in the same sequence building up a dimeric-like 4d-structure) are stable structures (see Fig. 1 in ref. ⁴). By aggregating the first β-strand in the start domain of one β-barrel to the ABIDG β-sheet of the other β-barrel, composing a big ABIDGY β-sheet (Y is the first β-strand). For instance, using the bicupin 1L3J (oxalate decarboxylase) as template, the low confidence level β-strand at position 26–33 (v in H29D30 avv) in type I NLPs corresponds to the first β-strand. Since this proceeds from both barrels they can build a stable structure (see Fig. 1 in ref. ⁴). The quaternary structure is related to the presence of interaction residues in the BID β-sheet of the cupin structure. These are present in the NLPs and would enable them to form dimers.Open in a separate windowFigure 2Alignment of type I NLP consensus, PpNPP1, MpNEP1 and MpNEP2. Solid line boxes are β-strands, double line boxes are α-helices. The sequence positions marked with asterisks (*) are possibly related to the differences in oligomeric properties between MpNEP1 and PpNPP1 with MpNEP2.     

Motor neuron disease and frontotemporal lobar degeneration: a tale of two disorders linked to TDP-43

Motor neuron disease (MND) is a neurodegenerative condition long thought to be associated only with motor weakness. Recent work now shows that cognitive difficulties are present in up to half of the patients with this disorder. About 5-10% of patients with MND have a frank dementia that resembles frontotemporal lobar degeneration (FTLD). Imaging studies show quantitative abnormalities that resemble FTLD. Moreover, biochemical studies of ubiquinated histopathologic abnormalities in MND and FTLD reveal identical inclusions of TDP-43. These findings underline a fundamental link between MND and FTLD. This paper reviews this body of work.     

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.     

Evaluation of the viability and germination of tempisque [<i>Sideroxylon capiri</i> (A.DC.) Pittier Sapotaceae]

The tempisque (Sideroxylon capiri) is a tree native to Mexico used by the rural population for housing construction, poles and hedges, as fuel (wood) and also for fodder and ornamental purposes, among others. It is considered an endangered species. In order to contribute to its preservation and sustainable management, it was considered important to determine the proportion of viable seeds, the loss of viability due to storage period and the germination process by applying pregerminative treatments. We found that freshly collected seeds showed 100% viability, which decreased to 0% after 5 months of storage. According to the cumulative germination significant differences between treatments (p≤0.01) were found. It was observed that seeds can accelerate their time of germination with the previous exposure of 24 h in water at room temperature. The soaking treatment in water for 24 h at room temperature obtained final germination of 55%, while with the control 39% was reached. Soaking in hydrogen peroxide and scarification were the treatments with lower germination percentage (33 and 23%, respectively). To get a higher percentage of germinated seeds in a short time, it is necessary to give a soaking treatment in water for 24 h before sowing.     

Regeneration of acetylcholinesterase in clonal neuroblastoma-glioma hybrid NG108-15 cells after soman inhibition: effect of glycyl-l-glutamine

Acetylcholinesterase (AChE) in the clonal NG108-15 cell line has been previously characterized. This cell line represents an in vitro system to study AChE regulation and effects of chemical compounds that may alter AChE activity. Recently, glycyl-L-glutamine (GLG) was demonstrated to function as a neurotrophic factor for maintenance of AChE content in cat denervated superior cervical ganglion cells. In the present study, regeneration of AChE activity in cultures of undifferentiated NG108-15 cells after soman inhibition was investigated in the presence and absence of GLG. Cells were treated with soman (5.5 × 10^–6 M) for 15 min and then washed to remove excess soman. Culture medium containing either GLG (10^–6, 10^–5, or 10^–4M) or glycyl-L-glutamic acid (10^–6 M) was added to cultures after soman treatment and remained in the medium until cell harvest. Cells were physically detached at various times after soman treatment and specific AChE activity was determined. After soman, AChE activity dramatically decreased to less than 1% of untreated cellular activity at 1 hr. AChE activity gradully increased after 5 hr, while untreated cell AChE activity was regained 20 hr after soman. The t_1/2 for AChE regeneration was approximately 10 hr. GLG did not increase the rate of AChE regeneration after soman inhibition. These results indicate that GLG is not a directly acting neurotrophic factor for AChE synthesis in NG108-15 cells after chemical AChE inactivation.Abbreviations AChE acetylcholinesterase - NG108-15 cell neuroblastoma-glioma 108-15 cell - DMEM Dulbecco's modified Eagles minimal essential medium - FBS fetal bovine serum - GLGA glycyl-L-glutamic acid - L-GA L-glutamic acid - GLG glycyl-L-glutamine - GD soman The opinions or assertions contained herein are the private views of the authors and are not to be construed as reflecting the view of the Department of the Army or the Department of the Army or the Department of Defense.     

Cantharidin analogues: synthesis and evaluation of growth inhibition in a panel of selected tumour cell lines

Diels-Alder addition of furans (furan, furfuryl alcohol, and 3-bromofuran) to maelic anhydride yields three distinct 5,6-dehydronorcantharidins. Hydrogenation of (4,10-dioxatricyclo[5.2.1.0]decane-3,5-dione) (4a), in dry ethanol affords the monoester (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic aid monoethyl ester) (6). Subsequent transesterification affords a series of monoesters (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid monomethyl ester (7)), 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid monopropyl ester (8), (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid monohexyl ester (9)) and differentially substituted diesters (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid 2-ethyl ester 3-isopropyl ester) (10), and (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid 2-ethyl ester 3-phenyl ester) (11). Analogues were firstly screened for their ability to inhibit protein phosphatases 1 (PP1) and 2A (PP2A) as the lead compounds cantharidin (1) and norcantharidin (2) are known PP1 and PP2A inhibitors. Only analogues 4a, 6-8 displayed good PP1 and PP2A inhibition (PP1 IC(50)'s=2.0, 2.96, 4.71, and 4.82 microM, respectively; PP2A IC(50)'s=0.2, 0.45, 0.41, and 0.47 microM, respectively). All analogues were also screened for their anti-cancer potential against a panel of tumour cell lines, HL60, L1210, SW480, WiDr, HT29, HCT116, A2780, ADDP, and 143B, producing GI(50) values ranging from 6 microM to >1000 microM. Analogues possessing good PP1 and/or PP2A inhibition also returned moderate to good anti-cancer activity. Analogues with substituents directly attached to the intact bicyclo[2.2.1]heptane skeleton were poor to moderate anti-cancer agents. This correlates well with their lack of PP1 or PP2A activity. Analogues capable of undergoing a facile ring opening of the anhydride or with a single carboxylate were good PP1 and PP2A inhibitors, largely correlating to the observed anti-cancer activity in all cases, except 11. Analogue 11, whist neither a PP1 nor a PP2A inhibitor shows anti-cancer activity comparable to 1 and 2. We believe that intracellular esterases generate the corresponding dicarboxylate, which is a potent PP1 and PP2A inhibitor, and that it is this species which is responsible for the observed anti-cancer activity.     

The effects of dihydropyridine calcium antagonists on heme biosynthesis in chick embryo liver cell culture

A variety of 1,4-dihydropyridine calcium antagonists were tested for porphyrinogenic activity in chick embryo liver cell culture. 3,5-Dimethoxycarbonyl-1,4-dihydro-2, 6-dimethyl-4-(ortho-nitrophenyl)pyridine (nifedipine) was shown to be a potent porphyrinogenic agent. This activity was shared by a number of related analogues, viz., the 4-phenyl, 4-(meta-nitrophenyl), 4-(para-nitrophenyl), 4-(ortho-methoxyphenyl), 4-(meta-trifluoromethylphenyl), and 4-(para-trifluoromethylphenyl) analogues and nitrendipine; nicardipine exhibited very weak activity. The porphyrinogenic potency of the 1,4-dihydropyridines did not parallel their calcium antagonist activity. Nifedipine did not exhibit ferrochelatase-lowering activity in chick embryo liver cell culture and uroporphyrin and heptacarboxylic acid porphyrin were the major porphyrins to accumulate. Nifedipine did not cause suicidal destruction of cytochrome P-450 in chick embryo hepatic microsomes. Because nifedipine possesses comparable porphyrinogenic activity to sodium secobarbital in chick embryo liver cell culture, caution is required if nifedipine or related drugs are administered to patients with hereditary hepatic porphyria.