Ser422 phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer’s Disease

Proteolytic truncation of microtubule associated human (h) Tau protein by caspase-3 at the carboxy (C) terminus has been linked to the pathogenesis of Alzheimer’s Disease (AD). This cleavage likely occurs between Asp⁴²¹↓Ser⁴²² leading to the formation of 421-mer truncated Tau protein which has been found to be present as aggregate in high level after phosphorylation in mortal AD brain tissue compared to normal. At least 50 phosphorylation sites involving Ser, Thr and Tyr residues have been identified or proposed in hTau and a selected number of them have been implicated in hTau aggregation following latter’s proteolytic truncation. Interestingly, it is further noted that Ser⁴²² residue present in the P1′ position of hTau caspase-3 cleavage region is a potential phosphorylation site. So we became interested to examine in vitro the effect of phospho-Ser⁴²² residue on hTau cleavage by caspase-3 which is a crucial upstream event associated with hTau self-assembly leading to AD pathogenesis. The goal of this project is to study in vitro the caspase-3 cleavage site of hTau protein and to examine the kinetics of this cleavage following Ser⁴²² phosphorylation and treatment with caspase-3 inhibitors. This is achieved by designing peptides from the sequence of hTau protein containing the proposed caspase-3 cleavage region. Peptides were designed from 441-mer major human Tau protein sequence that encompasses the proposed caspase-3 cleavage site [Asp⁴²¹↓Ser⁴²²]. Corresponding phospho-, dextro-Ser⁴²² and dextro-Asp⁴²¹ analogs were also designed. Peptides were synthesized by solid phase chemistry, purified and fully characterized by mass spectrometry. These were then incubated with recombinant caspase-3 enzyme under identical condition for digestion and analyzed for cleavage by mass spectrometry and RP-HPLC chromatograms. Our results indicated that while the control peptide is efficiently cleaved by caspase-3 at Asp⁴²¹↓Ser⁴²² site producing the expected N- and C-terminal fragment peptides, the corresponding phospho-Ser⁴²² peptide remained completely resistant to the cleavage. Substitution of Asp⁴²¹ by its dextro isoform also blocks peptide cleavage by caspase-3. However substitution of Ser⁴²² by its dextro isoform in the peptide did not affect the cleavage significantly. The above results were further confirmed by caspase-3 digestion experiment in the presence of varying amounts of caspase-3 inhibitor (Ac-DQVD-aldehyde) which was found to block this cleavage in a highly effective manner. Our results highlighted the crucial significance of Ser⁴²² phosphorylation and suggest that the kinase associated with this Ser-phosphorylation may protect Tau from aggregation. Thus specific promoters/activators of this kinase may find useful therapeutic benefits in arresting Tau truncation by caspase-3 and the progression of AD. In addition our data demonstrated that Tau-peptides where Ser⁴²² or Asp⁴²¹ are substituted by their respective dextro isomers, exhibit different cleavage kinetics by caspase-3 and this may have important implications in therapeutic intervention of Tau aggregation and associated AD.     

粘红酵母产L-苯丙氨酸解氨酶发酵培养基的优化

通过单因子和正交试验 ,对粘红酵母产 L -苯丙氨酸解氨酶 ( PAL )培养基进行优化 ,L-苯丙氨酸的积累浓度可以从 2 .0 g/1 0 0 ml提高到 3 .3 g/1 0 0 ml,最终得到了 L-苯丙氨酸解氨酶发酵的最适条件     

Salicylic acid biosynthesis is not from phenylalanine in Arabidopsis

The phytohormone salicylic acid(SA) regulates biotic and abiotic stress responses in plants. Two distinct biosynthetic pathways for SA have been well documented in plants: the isochorismate(IC)pathway in the chloroplast and the phenylalanine ammonia-lyase(PAL) pathway in the cytosol.However, there has been no solid evidence that the PAL pathway contributes to SA biosynthesis. Here,we report that feeding Arabidopsis thaliana with Ring-¹³C-labeled phenylalanine(¹³C₆     

Cavity spot of carrots: Interactions between the host and pathogen, related to the cell wall

This study investigates the structural aspects of cavity spot pathogenesis. Different Pythium spp. isolated from infected carrots, apples and melons were cultured on agar in Petri dishes and used for inoculation of uninfected carrots. Only slow-growing Pythium spp. (< 15 mm day^-1), such as P. violae and P. sulcatum caused cavity spot lesions. It is suggested that slow-growing species are able to penetrate, albeit slowly, into the plant tissue for 3 to 4 days before a hypersensitive reaction develops. Fast-growing species, however, did not cause lesions. Based on ultrastructural observations, we suggest that the following sequence of events occurs between the plant and the pathogen: The fungus infects the walls and grows for several days, during which time small amounts of wall-degrading enzymes are secreted. Phenylalanine ammonia lyase (PAL) activity and phenols increase linearly immediately upon inoculation. There was a lag phase of about 5 days before lignin began to increase linearly for about a month. Dissolution of wall components decreases the solute potential and water potential in the apoplast. Thus, water moves from the symplast into the apoplast, the turgor pressure gradually dissipates, and the cells shrink and eventually die.     

Differentiation of metabolic pathways in the umbel of Daucus carota

The extractable activities of PAL, chalcone synthase and chalcone isomerase of white and coloured petals of the inflorescence of the umbel of wild carr     

Identification of a phenylalanine ammonia-Iyase inactivating factor in harvested head lettuce (Lactuca sativa)

Exposing head lettuce ( Lactuca sativa L., crisphead or Iceberg type) leaf tissue to hormonal levels of ethylene (10 μl l⁻¹) at 5°C promotes the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and an increase in its activity. It also promotes the appearance of the postharvest physiological disorder called russet spotting (RS). Discontinuing ethylene exposure after 4 days resulted in a rapid decline in PAL activity which was delayed by treating excised midrib leaf tissue with actinornycin D or cycloheximide at 5°C. Only cycloheximide delayed the loss of PAL activity in tissue that was transferred from 5 to 15°C. Activity of PAL from Rhodolorula glutinis was. slowly lost during incubation in buffer alone, but there was a logarithmic decline in its activity over time when it was incubated with aliquots of the resuspended 10000 g pellet from homogenized, lettuce tissue affected with RS. The in vitro loss in PAL activity was 9–fold higher in extracts from lettuce showing RS symptoms than from control lettuce, boiled samples or the buffer control. The PAL-inactivating factor isolated from lettuce affected with RS had a pH optimum around 8.0. It appears that the rapid loss in PAL activity after the discontinuation of exposure to ethylene is dependent on the de novo synthesis of a PAL-inactivating factor.     

Induction of total phenolics and defence-related enzymes during β-aminobutyric acid-induced resistance in Brassica carinata against Alternaria blight

Exogenous foliar application of β-aminobutyric acid (BABA) led to a significant reduction in disease severity in Brassica carinata caused by Alternaria brassicae. To get a better insight about changes in defence-related enzymes like phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), isoform analysis of superoxide dismutase (SOD) and peroxidase (POX) were studied. BABA-treated plants showed a significant increase in PAL, PPO enzyme activities and total phenolic content in response to pathogen inoculation. However, isoform analysis of SOD and POX revealed no change in isoform number but a quantitative change in activity was observed in response to pathogen.     

ATP-NGF-complex, but not NGF, is the neuroprotective ligand

We have shown previously that nerve growth factor (NGF) requires only low nanomolar ATP concentrations in the cell culture medium to protect cortical rat neurons (CRN) from cellular damage induced by staurosporine (STS). We have also demonstrated before that NGF and other growth factors form stable non-covalent complexes with ATP. Here we demonstrated that 8N₁ATP–NGF, but not NGF, protected CRN against damage. The photo-reactive ATP derivative 8N₃ATP was incubated with NGF and was trapped in its position by UV irradiation forming a covalent bond. The cross-link with a molar ratio of 1:1 (8N₁ATP:NGF) was confirmed by mass spectrometry. Circular dichroism experiments revealed that 8N₁ATP altered the secondary structure of NGF in the same way as ATP did. Covalently bound 8N₁ATP–NGF was shown to be stable in the presence of the ATP-hydrolyzing enzyme alkaline phosphatase while the non-covalent ATP–NGF-complex dissociated with the removal of free ATP from the solution. 8N₁ATP–NGF protected CRN against damage by STS independently of free ATP in the culture medium. These results suggest that the ATP–NGF-complex, but not NGF, is the active ligand.