Tumor necrosis factor alpha-induced desumoylation and cytoplasmic translocation of homeodomain-interacting protein kinase 1 are critical for apoptosis signal-regulating kinase 1-JNK/p38 activation

The apoptosis signal-regulating kinase 1 (ASK1)-JNK/p38 signaling pathway is pivotal component in cell apoptosis and can be activated by a variety of death stimuli including tumor necrosis factor (TNF) alpha and oxidative stress (reactive oxygen species). However, the mechanism for ASK1 activation is not fully understood. We have recently identified ASK1-interacting protein (AIP1) as novel signal transducer in TNFalpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. In the present study, we employed yeast two-hybrid system using the N-terminal domain of AIP1 as bait and identified homeodomain-interacting protein kinase 1 (HIPK1) as an AIP1-associated protein. Interestingly, we showed that TNFalpha induced HIPK1 desumoylation concomitant with a translocation from nucleus to cytoplasm at 15 min followed by a return to nucleus by 60 min. The kinetics of HIPK1 translocation correlates with those of stress-induced ASK1-JNK/P38 activation. A specific JNK inhibitor blocked the reverse but not the initial translocation of HIPK1, suggesting that the initial translocation is an upstream event of ASK1-JNK/p38 signaling and JNK activation regulates the reverse translocation as a feedback mechanism. Consistently, expression of HIPK1 increased, whereas expression of a kinase-inactive form (HIPK1-D315N) or small interference RNA of HIPK1 decreased stress-induced ASK1-JNK/P38 activation without effects on IKK-NF-kappaB signaling. Moreover, a sumoylation-defective mutant of HIPK1 (KR5) localizes to the cytoplasm and is constitutively active in ASK1-JNK/P38 activation. Furthermore, HIPK1-KR5 induces dissociation of ASK1 from its inhibitors 14-3-3 and thioredoxin and synergizes with AIP1 to induce ASK1 activation. Our study suggests that TNFalpha-induced desumoylation and cytoplasmic translocation of HIPK1 are critical in TNFalpha-induced ASK1-JNK/p38 activation.     

Microtubule regulation of N-methyl-D-aspartate receptor channels in neurons

N-Methyl-D-aspartate (NMDA) receptors (NMDARs), which play a key role in synaptic plasticity, are dynamically regulated by many signaling molecules and scaffolding proteins. Although actin cytoskeleton has been implicated in regulating NMDAR stability in synaptic membrane, the role of microtubules in regulating NMDAR trafficking and function is largely unclear. Here we show that microtubule-depolymerizing agents inhibited NMDA receptor-mediated ionic and synaptic currents in cortical pyramidal neurons. This effect was Ca(2+)-independent, required GTP, and was more prominent in the presence of high NMDA concentrations. The NR2B subunit-containing NMDA receptor was the primary target of microtubules. The effect of microtubule depolymerizers on NMDAR currents was blocked by cellular knockdown of the kinesin motor protein KIF17, which transports NR2B-containing vesicles along microtubule in neuronal dendrites. Neuromodulators that can stabilize microtubules, such as brain-derived neurotrophic factor, significantly attenuated the microtubule depolymerizer-induced reduction of NMDAR currents. Moreover, immunocytochemical studies show that microtubule depolymerizers decreased the number of surface NR2B subunits on dendrites, which was prevented by the microtubule stabilizer. Taken together, these results suggest that interfering with microtubule assembly suppresses NMDAR function through a mechanism dependent on kinesin-based dendritic transport of NMDA receptors.     

Ab initio molecular dynamics simulations of beta-D-glucose and beta-D-xylose degradation mechanisms in acidic aqueous solution

Ab initio molecular dynamics simulations were employed to investigate, with explicit solvent water molecules, beta-D-glucose and beta-D-xylose degradation mechanisms in acidic media. The rate-limiting step in sugar degradation was found to be protonation of the hydroxyl groups on the sugar ring. We found that the structure of water molecules plays a significant role in the acidic sugar degradation pathways. Firstly, a water molecule competes with the hydroxyl group on the sugar ring for protons. Secondly, water forms hydrogen bonds with the hydroxyl groups on the sugar rings, thus weakening the C-C and C-O bonds (each to a different degree). Note that the reaction pathways could be altered due to the change of relative stability of the C-C and C-O bonds. Thirdly, water molecules that are hydrogen-bonded to sugar hydroxyls could easily extract a proton from the reaction intermediate, terminating the reaction. Indeed, the sugar degradation pathway is complex due to multiple protonation probabilities and the surrounding water structure. Our experimental data support multiple sugar acidic degradation pathways.     

Syntabulin-mediated anterograde transport of mitochondria along neuronal processes

In neurons, proper distribution of mitochondria in axons and at synapses is critical for neurotransmission, synaptic plasticity, and axonal outgrowth. However, mechanisms underlying mitochondrial trafficking throughout the long neuronal processes have remained elusive. Here, we report that syntabulin plays a critical role in mitochondrial trafficking in neurons. Syntabulin is a peripheral membrane-associated protein that targets to mitochondria through its carboxyl-terminal tail. Using real-time imaging in living cultured neurons, we demonstrate that a significant fraction of syntabulin colocalizes and co-migrates with mitochondria along neuronal processes. Knockdown of syntabulin expression with targeted small interfering RNA or interference with the syntabulin-kinesin-1 heavy chain interaction reduces mitochondrial density within axonal processes by impairing anterograde movement of mitochondria. These findings collectively suggest that syntabulin acts as a linker molecule that is capable of attaching mitochondrial organelles to the microtubule-based motor kinesin-1, and in turn, contributes to anterograde trafficking of mitochondria to neuronal processes.     

Identification of single-chain antibody fragments specific against SARS-associated coronavirus from phage-displayed antibody library

To develop early diagnostic reagents, effective vaccines, and even drugs against SARS-associated coronavirus (SARS-CoV), the human single fold single-chain antibody fragments, (scFv) libraries I+J (Tomlinson I+J) were used to identify novel scFvs, which can specifically bind to SARS-CoV. Interestingly, two scFvs (B5 and B9) exhibited higher binding specificity to SARS-CoV with the OD(450) value 0.608 and 0.545, respectively, and their coding sequences shared the identical sequence composed of V(H) gene (351bp) and V(L) gene (327bp), so the two scFvs were uniformly named as SA59B and chosen for further analysis. SA59B scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The soluble 30kDa SA59B scFv-antibody was verified in SDS-PAGE and Western-blot. The purified SA59B scFv-antibody was labeled with HRP by the glutaraldehyde method, and the concentration of HRP and SA59B scFv-antibody in the SA59B-HRP solution reached 2.4 and 2.28mg/ml, respectively. Then, the binding ability of SA59B-HRP to SARS-CoV was evaluated by ELISA with S/N of 11.6, indicating higher binding specificity between them. Finally, both the SA59B sequence specificity and its application for diagnosis, prophylaxis or therapy of SARS were discussed.     

Clinical evaluation and mitochondrial DNA sequence analysis in two Chinese families with aminoglycoside-induced and non-syndromic hearing loss

We report here the clinical, genetic, and molecular characterization of two Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects. Penetrances of hearing loss in BJ105 and BJ106 pedigrees are 67% and 33%, respectively. In particular, three of 10 affected matrilineal relatives of BJ105 pedigree had aminoglycoside-induced hearing loss, while seven affected matrilineal relatives in BJ105 pedigree and six affected matrilineal relatives in BJ106 pedigree did not have a history of exposure to aminoglycosides. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mtDNA variants belonging to haplogroups F3 and M7b. These variants showed no evolutionary conservation, implying that mitochondrial haplotype may not play a significant role in the phenotypic expression of the A1555G mutation in these Chinese pedigrees. However, aminoglycosides and nuclear backgrounds appear to be major modifier factors for the phenotypic manifestation of the A1555G mutation in these Chinese families.     

Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active

The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide-linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design.     

Theaflavin derivatives in black tea and catechin derivatives in green tea inhibit HIV-1 entry by targeting gp41

Theaflavin derivatives and catechin derivatives are the major polyphenols in black tea and green tea, respectively. Several tea polyphenols, especially those with galloyl moiety, can inhibit HIV-1 replication with multiple mechanisms of action. Here we showed that the theaflavin derivatives had more potent anti-HIV-1 activity than catechin derivatives. These tea polyphenols could inhibit HIV-1 entry into target cells by blocking HIV-1 envelope glycoprotein-mediated membrane fusion. The fusion inhibitory activity of the tea polyphenols was correlated with their ability to block the formation of the gp41 six-helix bundle, a fusion-active core conformation. Computer-aided molecular docking analyses indicate that these tea polyphenols, theaflavin-3,3'-digallate (TF3) as an example, may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil formed by the N-terminal heptad repeats of gp41. These results indicate that tea, especially black tea, may be used as a source of anti-HIV agents and theaflavin derivatives may be applied as lead compounds for developing HIV-1 entry inhibitors targeting gp41.     

Nitration and oligomerization of tau induced by peroxynitrite inhibit its microtubule-binding activity

Abnormally nitrated tau has been found recently in the neurofibrillary tangles of Alzheimer's disease (AD). However, whether and how nitration of tau is involved in AD pathology is not known. Herein, we found that in vitro incubation of peroxynitrite with recombinant tau resulted in nitration and oligomerization of tau in a dosage-dependent manner. Moreover, the nitrated tau showed a significantly decreased binding activity to taxol-stabilized microtubulesin in vitro. Further study demonstrated that peroxynitrite also induced tau nitration in neuroblastoma N2a cell line, and the nitrated tau was accumulated in the cells. We conclude that abnormal nitration of tau contributes to the impaired biological activity of tau in binding to the microtubules and the aggregation of tau, implying a novel mechanism responsible for the neurodegeneration seen in AD brain.     

Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition.