Identification of hydrophobic interactions between proteins and lipids: free fatty acids activate phospholipase C delta1 via allosterism

Lipids are well recognized ligands that bind to proteins in a specific manner and regulate their function. Most attention has been placed on the headgroup of phospholipids, and little is known about the role of the acyl chains in mediating any effects of lipids on proteins. In this report, free fatty acids (FFA) were found to bind and activate phospholipase C delta1(PLC delta1). The unsaturated FFA arachidonic acid (AA) and oleic acid were able to stimulate PLC delta1 up to 30-fold in a dose-dependent manner. The saturated FFA stearic acid and palmitic acid were less efficacious than unsaturated FFA, activating the enzyme up to 8-fold. The mechanism of activation appears to be due to a change in K(m) for substrate; 50 microM arachidonate reduced the K(m) for the soluble PLC substrate diC(4)PI from 1.7 +/- 0.6 mM to 0.24 +/- 0.04 mM (7-fold reduction). V(max) was not significantly altered. PLC delta1 bound to sucrose-loaded vesicles that contained AA in a concentration-dependent manner. A fragment of PLC delta1 that encompasses the EF-hand domain also bound to micelles containing AA using nondenaturing PAGE. This same fragment also inhibited AA activation of PLC delta1 in a competition assay. These results suggest that the function of the EF-hand domain of PLC delta1 is to bind lipid and to allosterically regulate catalysis. These results also suggest that esterified and nonesterified fatty acids can bind to and regulate protein function, identifying a functional role for hydrophobic interactions between lipids and proteins.     

Genomewide association analysis of human narcolepsy and a new resistance gene

Human narcolepsy is a hypersomnia that is affected by multiple genetic and environmental factors. One genetic factor strongly associated with narcolepsy is the HLA-DRB1*1501-DQB1*0602 haplotype in the human leukocyte antigen region on chromosome 6, whereas the other genetic factors are not clear. To discover additional candidate regions for susceptibility or resistance to human narcolepsy, we performed a genomewide association study, using 23,244 microsatellite markers. Two rounds of screening with the use of pooled DNAs yielded 96 microsatellite markers (including 16 markers on chromosome 6) with significantly different estimated frequencies in case and control pools. Markers not located on chromosome 6 were evaluated by the individual typing of 95 cases and 95 controls; 30 markers still showed significant associations. A strong association was displayed by a marker on chromosome 21 (21q22.3). The surrounding region was subjected to high-density association mapping with 14 additional microsatellite markers and 74 SNPs. One microsatellite marker (D21S0012m) and two SNPs (rs13048981 and rs13046884) showed strong associations (P < .0005; odds ratios 0.19-0.33). These polymorphisms were in a strong linkage disequilibrium, and no other polymorphism in the region showed a stronger association with narcolepsy. The region contains three predicted genes--NLC1-A, NLC1-B, and NLC1-C--tentatively named "narcolepsy candidate-region 1 genes," and NLC1-A and NLC1-C were expressed in human hypothalamus. Reporter-gene assays showed that the marker D21S0012m in the promoter region and the SNP rs13046884 in the intron of NLC1-A significantly affected expression levels. Therefore, NLC1-A is considered to be a new resistance gene for human narcolepsy.     

Post-translational Modifications of Recombinant Human Lysyl Oxidase-like 2 (rhLOXL2) Secreted from Drosophila S2 Cells

Human lysyl oxidase-like 2 (hLOXL2) is highly up-regulated in metastatic breast cancer cells and tissues and induces epithelial-to-mesenchymal transition, the first step of metastasis/invasion. hloxl2 encodes four N-terminal scavenger receptor cysteine-rich domains and the highly conserved C-terminal lysyl oxidase (LOX) catalytic domain. Here, we assessed the extent of the post-translational modifications of hLOXL2 using truncated recombinant proteins produced in Drosophila S2 cells. The recombinant proteins are soluble, in contrast to LOX, which is consistently reported to require 2–6 m urea for solubilization. The recombinant proteins also show activity in tropoelastin oxidation. After phenylhydrazine derivatization and trypsin digestion, we used mass spectrometry to identify peptides containing the derivatized lysine tyrosylquinone cross-link at Lys-653 and Tyr-689, as well as N-linked glycans at Asn-455 and Asn-644. Disruption of N-glycosylation by site-directed mutagenesis or tunicamycin treatment completely inhibited secretion so that only small quantities of inclusion bodies were detected. The N-glycosylation site at Asn-644 in the LOX catalytic domain is not conserved in human LOX (hLOX), although the LOX catalytic domain of hLOX shares ∼50% identity and ∼70% homology with hLOXL2. The catalytic domain of hLOX was not secreted from S2 cells using the same expression system. These results suggest that the N-glycan at Asn-644 of hLOXL2 enhances the solubility and stability of the LOX catalytic domain.     

Evolutionary Analysis of Classical HLA Class I and II Genes Suggests That Recent Positive Selection Acted on DPB1*04∶01 in Japanese Population

The human leukocyte antigen (HLA) genes exhibit the highest degree of polymorphism in the human genome. This high degree of variation at classical HLA class I and class II loci has been maintained by balancing selection for a long evolutionary time. However, little is known about recent positive selection acting on specific HLA alleles in a local population. To detect the signature of recent positive selection, we genotyped six HLA loci, HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1 in 418 Japanese subjects, and then assessed the haplotype homozygosity (HH) of each HLA allele. There were 120 HLA alleles across the six loci. Among the 80 HLA alleles with frequencies of more than 1%, DPB1*04∶01, which had a frequency of 6.1%, showed exceptionally high HH (0.53). This finding raises the possibility that recent positive selection has acted on DPB1*04∶01. The DPB1*04∶01 allele, which was present in the most common 6-locus HLA haplotype (4.4%), A*33∶03-C*14∶03-B*44∶03-DRB1*13∶02-DQB1*06∶04-DPB1*04∶01, seems to have flowed from the Korean peninsula to the Japanese archipelago in the Yayoi period. A stochastic simulation approach indicated that the strong linkage disequilibrium between DQB1*06∶04 and DPB1*04∶01 observed in Japanese cannot be explained without positive selection favoring DPB1*04∶01. The selection coefficient of DPB1*04∶01 was estimated as 0.041 (95% credible interval 0.021–0.077). Our results suggest that DPB1*04∶01 has recently undergone strong positive selection in Japanese population.     

A novel recombinant system for functional expression of myonecrotic snake phospholipase A(2) in Escherichia coli using a new fusion affinity tag

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A₂ ([Lys⁴⁹]PLA₂), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as α-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for α-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity.     

Reduction of methamphetamine-induced sensitization and reward in matrix metalloproteinase-2 and -9-deficient mice

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) function to remodel the pericellular environment. Their activation and regulation are associated with synaptic physiology and pathology. Here, we investigated whether MMP-2 and MMP-9 are involved in the rewarding effects of and sensitization to methamphetamine (METH) in animals, in which the remodelling of neural circuits may play a crucial role. Repeated METH treatment induced behavioural sensitization, which was accompanied by an increase in MMP-2 and MMP-9 activity in the brain. In MMP-2- and MMP-9-deficient mice [MMP-2-(-/-) and MMP-9-(-/-)], METH-induced behavioural sensitization and conditioned place preference, a measure of the rewarding effect, as well as METH-increased dopamine release in the nucleus accumbens (NAc) were attenuated compared with those in wild-type mice. In contrast, infusion of purified human MMP-2 into the NAc significantly potentiated the METH-increased dopamine release. The [(3)H]dopamine uptake into striatal synaptosomes was reduced in wild-type mice after repeated METH treatment, but METH-induced changes in [(3)H]dopamine uptake were significantly attenuated in MMP-2-(-/-) and MMP-9-(-/-) mice. These results suggest that both MMP-2 and MMP-9 play a crucial role in METH-induced behavioural sensitization and reward by regulating METH-induced dopamine release and uptake in the NAc.     

Role of matrix metalloproteinase and tissue inhibitor of MMP in methamphetamine-induced behavioral sensitization and reward: implications for dopamine receptor down-regulation and dopamine release

Matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) function to remodel the pericellular environment. We have demonstrated that methamphetamine (METH)-induced behavioral sensitization and reward were markedly attenuated in MMP-2- and MMP-9 deficient [MMP-2-(-/-) and MMP-9-(-/-)] mice compared with those in wild-type mice, suggesting that METH-induced expression of MMP-2 and MMP-9 in the brain plays a role in the development of METH-induced sensitization and reward. In the present study, we investigated the changes in TIMP-2 expression in the brain after repeated METH treatment. Furthermore, we studied a role of MMP/TIMP system in METH-induced behavioral changes and dopamine neurotransmission. Repeated METH treatment induced behavioral sensitization, which was accompanied by an increase in TIMP-2 expression. Antisense TIMP-2 oligonucleotide (TIMP-AS) treatment enhanced the sensitization, which was associated with the potentiation of METH-induced dopamine release in the nucleus accumbens (NAc). On the other hand, MMP-2/-9 inhibitors blocked the METH-induced behavioral sensitization and conditioned place preference, a measure of the rewarding effect, and reduced the METH-increased dopamine release in the NAc. Dopamine receptor agonist-stimulated [(35)S]GTPgammaS binding was reduced in the frontal cortex of sensitized rats. TIMP-AS treatment potentiated, while MMP-2/-9 inhibitor attenuated, the reduction of dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding. Repeated METH treatment also reduced dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding in wild-type mice, but such changes were significantly attenuated in MMP-2-(-/-) and MMP-9-(-/-) mice. These results suggest that the MMP/TIMP system is involved in METH-induced behavioral sensitization and reward, by regulating dopamine release and receptor signaling.