ATP-induced focal adhesion kinase activity is negatively modulated by phospholipase D2 in PC12 cells

Extracellular ATP has been known to modulate various cellular responses including mitogenesis, secretion and morphogenic activity in neuronal cells. In the ATP-induced morphogenic activity, focal adhesion kinase(s) such as Fak have been suggested to play a critical role. Binding of ATP to its specific cell surface receptor in PC12 cells induces phospholipase D (PLD) activity. However, the role of PLD on ATP-induced Fak activation in PC12 cells remains unclear. In this study, we investigated the role of PLD on the ATP-induced Fak activation and paxillin phosphorylation using two established cell lines: wild type PLD2- and lipase-inactive mutant PLD2-inducible PC12 cells. Stimulation of cells with ATP caused PLD2 activation via classical protein kinase C activation. ATP also induced Fak activation, and paxillin phosphorylation, and were dramatically reduced by wild type PLD2 overexpression but not by lipase-inactive mutant PLD2 overexpression. When the PC12 cells were pretreated with propranolol, a specific inhibitor for phosphatidic acid phosphohydrolase resulting in the accumulation of PA, ATP-induced Fak activation and paxillin phosphorylation were also reduced. We found that inhibition of tyrosine phosphatases by pervanadate completely blocked PLD2-dependent Fak and paxillin dephosphorylation. Taken together, we suggest that PLD2 activity might play a negative role in ATP-induced Fak and paxillin phosphorylation possibly through tyrosine phosphatases.     

Function of ROC4 in the Efficient Repair of Photodamaged Photosystem II in Arabidopsis†

ROC4 is the only cyclophilin in the chloroplast stroma. Here, we used the T‐DNA knockout mutant of roc4 to study the physiological role of ROC4 in vivo in Arabidopsis thaliana. Our results showed that ROC4 is not required for the biogenesis and functional operation of photosystem II (PSII). However, growth in greenhouse and PSII activity, as detected by photoinhibition measurements showed increased sensitivity to high light irradiance in the mutant. In the presence of chloroplast protein synthesis inhibitor lincomycin, which blocks de novo protein synthesis and thus the repair of PSII, wild‐type and mutant plants showed a similar extent of inactivation of PSII activity. The recovery of PSII activity in roc4 leaves from photoinhibition is also impaired compared with that of wild‐type plants. Immunoblot analysis showed that the degradation of PSII reaction center proteins occurred at a similar rate in the presence of lincomycin in wild‐type and mutant plants. Thus, these results suggest that ROC4 functions in the repair of photodamaged PSII.     

Contribution of LHC II complex to the electric properties of thylakoid membranes: an electric light scattering study of Chl b-less barley mutant

Electric light scattering measurements demonstrate a strong decline in the permanent electric dipole moment and electric polarizability of both thylakoid membranes and photosystem II-enriched particles of the Chlorina f2 mutant which has severely reduced levels of light-harvesting chlorophyll a/b-binding proteins compared to the wild type barley chloroplasts. The shift in the electric polarizability relaxation to higher frequencies in thylakoids and photosystem II particles from Chlorina f2 reflects higher mobility of the interfacial charges of the mutant than that of the wild type membranes. The experimental data strongly suggest that the major light-harvesting complex of photosystem II directly contribute to the electric properties of thylakoid membranes.     

Derepressed 2-deoxyglucose-resistant mutants of Aspergillus niger with altered hexokinase and acid phosphatase activity in hyperproduction of β-fructofuranosidase

Aspergillus niger NRRL330 produces extracellular β-fructofuranosidase (Ffase), and its production is subject to repression by hexoses in the medium. After ultraviolet mutagenization and selection, seven derepressed mutants resistant to 2-deoxyglucose (2-DG) were isolated on Czapek’s minimal medium containing glycerol. One of the mutants, designated DGRA-1, produced higher levels of Ffase. A considerable difference occurred in the mutants with reference to hexokinase and intracellular acid phosphatase activities. The hexokinase activity of the mutant DGRA-1 (0.69 U/mg) was 1.8-fold higher than the wild type (0.38U/mg). Intracellular acid phosphatase activity of the mutant DGRA-1 (0.83 U/g of mycelia) was twofold higher than that of the wild type (0.42U/g of mycelia), suggesting that phosphorylation and dephosphorylation steps could attribute to the 2-DG resistance of A. niger. However, additional mutations could account for the increased production of Ffase in the mutant DGRA-1.     

Impaired phosphorylation and mis-localization of Bub1 and BubR1 are responsible for the defective mitotic checkpoint function in Brca2-mutant thymic lymphomas

Breast cancer susceptibility gene, BRCA2, is a tumor suppressor and individuals who inherit one defected copy of BRCA2 allele experience early onset breast cancer or ovarian cancer accompanied by the loss of the wild type allele. Mouse model for Brca2 mutation shows growth retardation and paradoxical occurrence of thymic lymphomas. Thymic lymphomas from Brca2-mutant mice harbor mutations in p53, Bub1, and BubR1, which function as mitotic checkpoint proteins. Therefore, interplay between Brca2 and mitotic checkpoint has been suggested in the maintenance of genetic fidelity, although it has not been assessed whether the unique mutations in Bub1 and BubR1 found in Brca2-mutant mice are responsible for the abolishment of mitotic checkpoint function. This report demonstrates that Bub1 and BubR1 mutant proteins from Brca2-/- thymic lymphomas have defects in the phosphorylation and kinetochore localization after spindle damage. Thus, the mutations of Bub1 and BubR1 found in Brca2- mutant mice indeed are responsible for the chromosome instability in Brca2-mutated tumors.     

Photosystem II activity and turnover of the D1 protein are impaired in the psbA Y112L mutant of Synechocystis PCC6803 sp.

Site-directed psbA mutants at the tyrosine Y112 position have been generated in Synechocystis PCC6803 cells. The mutation Y112F does not affect photosystem II (PSII) activity as compared with control 4 delta 1K cells. However, the Y112L mutant exhibits a photosynthetically impaired phenotype. PSII activity is not detectable in this mutant when grown at 30 mumol photons m-2 s-1, while low levels of the D1 and D2 proteins and oxygen evolution activity are present in the mutant cells grown at a low light intensity (0.5-1 mumol m-2 s-1). The recombination of the QB-/S2,3 states of PSII in the Y112L mutant cells as detected by thermoluminescence (TL) is altered. The TL signal emission maximum of these cells due to charge recombination of the S2,3/QB- occurs at 20 degrees C as compared to 35-40 degrees C for the wild-type cells, indicating a possible change in the S2,3/Yz equilibrium. The Y112L mutant cells are rapidly photoinactivated and impaired in the recovery of the PSII activity. These results suggest that replacement of the aromatic residue at position Y112 by a hydrophobic amino acid may alter the function of the donor-side activity and affects the degradation and replacement of the PSII core proteins.     

Accelerated Degradation of the D2 Protein of Photosystem II Under Ultraviolet Radiation

The D2 protein of photosystem II is relatively stable in vivo under photosynthetic active radiation, but its degradation accelerates under UVB radiation. Little is known about accelerated D2 protein degradation. We characterized wavelength dependence and sensitivity toward photosystem II inhibitors. The in vivo D2 degradation spectrum resembles the pattern for the rapidly turning over D1 protein of photosystem II, with rates being maximal in the UVB region. We propose that D2 degradation, like D1 degradation, is activated by distinct photosensitizers in the UVB and visible regions of the spectrum. In both wavelength regions, photosystem II inhibitors that are known to be targeted to the D1 protein affect D2 degradation. This suggests that degradation of the two proteins is coupled, D2 degradation being influenced by events occurring at the Q_B niche on the D1 protein.     

Amplified Degradation of Photosystem II D1 and D2 Proteins under a Mixture of Photosynthetically Active Radiation and UVB Radiation: Dependence on Redox Status of Photosystem II

Abstract— Plants exposed to a mixture of photosynthetically active radiation (PAR) and UVB radiation exhibit a marked boost in degradation of the D1 and D2 photosysteni II (PS II) reaction center proteins beyond that predicted by the sum of rates in PAR and UVB alone (amplified degradation). Becausee degradation driven by visible or UVB radiation alone is uncoupled from PS II redox status, it was therefore assumed that the mixed-light-amplified component of degradation would behave similarly. Surprisingly, amplified degradation proved to be coupled tightly to the redox status of PS II. We show that inactivation of the PS II water oxidation by heat shock or oxidation of the plastosemiquinone (Q_A-) by silicomolybdate nullifies only the amplified component of degradation but not the basic rates of degradation under PAR or UVB alone. The data are interpreted to indicate that formation of plastosemiquinone or an active water-oxidizing Mn₄ cluster, is the UVB chromophore involved in amplified degradation of the D1 and D2 proteins. Furthermore, accumulation of Q_A-by 3-(3,4-dichlorophenyl)-1,1-dimethylurea or 2-bromo-3-methyl-6-isopropyl-4-nitrophenol stimulated the mixed-light-amplified degradation component. Thus, amplified degradation of the D1 and D2 proteins in mixed radiance of PAR plus UVB (which simulates naturally occurring radiance) proceeds by a mechanism clearly distinct from that involved in degradation under PAR or UVB alone.     

The Lack of Lutein Accelerates the Extent of Light‐induced Bleaching of Photosynthetic Pigments in Thylakoid Membranes of Arabidopsis thaliana

The high light‐induced bleaching of photosynthetic pigments and the degradation of proteins of light‐harvesting complexes of PSI and PSII were investigated in isolated thylakoid membranes of Arabidopsis thaliana, wt and lutein‐deficient mutant lut2, with the aim of unraveling the role of lutein for the degree of bleaching and degradation. By the means of absorption spectroscopy and western blot analysis, we show that the lack of lutein leads to a higher extent of pigment photobleaching and protein degradation in mutant thylakoid membranes in comparison with wt. The highest extent of bleaching is suffered by chlorophyll a and carotenoids, while chlorophyll b is bleached in lut2 thylakoids during long periods at high illumination. The high light‐induced degradation of Lhca1, Lhcb2 proteins and PsbS was followed and it is shown that Lhca1 is more damaged than Lhcb2. The degradation of analyzed proteins is more pronounced in lut2 mutant thylakoid membranes. The lack of lutein influences the high light‐induced alterations in organization of pigment–protein complexes as revealed by 77 K fluorescence.     

Atomistic simulation reveals structural mechanisms underlying D614G spike glycoprotein-enhanced fitness in SARS-COV-2

D614G spike glycoprotein (sgp) mutation in rapidly spreading severe acute respiratory syndrome coronavirus-2 (SARS-COV-2) is associated with enhanced fitness and higher transmissibility in new cases of COVID-19 but the underlying mechanism is unknown. Here, using atomistic simulation, a plausible mechanism has been delineated. In G614 sgp but not wild type, increased D(G)614-T859 Cα-distance within 65 ns is interpreted as S1/S2 protomer dissociation. Overall, ACE2-binding, post-fusion core, open-state and sub-optimal antibody-binding conformations were preferentially sampled by the G614 mutant, but not wild type. Furthermore, in the wild type, only one of the three sgp chains has optimal communication route between residue 614 and the receptor-binding domain (RBD); whereas, two of the three chains communicated directly in G614 mutant. These data provide evidence that D614G sgp mutant is more available for receptor binding, cellular invasion and reduced antibody interaction; thus, providing framework for enhanced fitness and higher transmissibility in D614G SARS-COV-2 mutant.     

REGULATION OF PROTEIN PHOSPHORYLATION BY PHYTOCHROME IN Sorghum bicolor

Abstract— In vitro phosphorylation of some polypeptides was affected in extracts obtained from 5-and 6-day-old plants irradiated with 5 min of red light. The phosphorylation of 55 kDa polypeptide in both 5- and 6-day-old plants, a 60 kDa, and 76 kDa polypeptide in 6-day-old plants and 70 kDa, 67 kDa polypeptide in 5-day-old plants was stimulated by red light. This effect was reversible by far-red light. The extent of stimulation by red light and reversal by far-red light varied for different polypeptides. No differential effect of red and far-red light was seen on the phosphorylation of 94 and 40 kDa polypeptides. In fact, phosphorylation of 94 kDa polypeptide in 6-day-old plants decreased on red light irradiation. These results show that the phosphorylation or dephosphorylation of some proteins is affected by phytochrome and the effect of light is also dependent on the age of the plant.     

Thermal stabilization of tissues and the preservation of protein phosphorylation states for two‐dimensional gel electrophoresis

2‐DE is typically capable of discriminating proteins differing by a single phosphorylation or dephosphorylation event. However, a reliable representation of protein phosphorylation states as they occur in vivo requires that both phosphatases and kinases are rapidly and completely inactivated. Thermal stabilization of mouse cerebral cortex homogenates effectively inactivated these enzymes, as evidenced by comparison with unstabilized tissues where abscissal pI shifts were a common feature in 2‐D gels. Of the 588 matched proteins separated on 2‐D gels comparing stabilized and unstabilized tissues, 53 proteins exhibited greater than twofold differences in spot volume (ANOVA, p<0.05). Phosphoprotein‐specific staining was corroborated by the identification of 16 phosphoproteins by nano‐LC MS/MS and phosphotyrosine kinase activity assay.     

Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base‐Modified Nucleosides

A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild‐type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8‐aza‐7‐deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α‐D ‐pentofuranose‐1‐phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2‐deoxy‐α‐D ‐ribofuranose‐1‐phosphate in the trans‐2‐deoxyribosylation reaction. 5‐Aza‐7‐deazaguanine manifested excellent substrate activity for both enzymes, 8‐amino‐7‐thiaguanine and 2‐aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2‐amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1‐ and unusual N2‐glycosides, respectively. 9‐Deaza‐5‐iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9‐deazaxanthine and its 2′‐deoxyriboside are weak inhibitors.     

RESPONSE OF THE WILD-TYPE and HIGH LIGHT-TOLERANT MUTANT OF Anacystis nidulans AGAINST PHOTOOXIDATIVE DAMAGE: DIFFERENTIAL MECHANISM OF HIGH LIGHT TOLERANCE

Abstract— A high light-tolerant mutant of Anacystis was able to tolerate about three-fold higher light energy irradiance (30 W m^-2) than the wild type (10 W m^-2). The loss of sulfhydryl content and rate of lipid peroxidation in the wild-type cells is lower than in the mutant cells at high light irradiance. This phenomenon in the wild type is probably due to high light-induced severe photoinhibitory conditions resulting in a decreased rate of O₂ evolution. Results on the bleaching of the N, N '-dimethyl- p -nitrosoaniline at high light irradiance show a higher rate of bleaching in the wild-type than in the mutant cells. Further, results on the rate of N, N '-dimethyl- p -nitrosoani)ine bleaching in the presence of radical scavengers like sodium azide, histidine and sodium formate (10 m M , each) suggest that singlet oxygen is the predominant oxygen species produced in both the wild-type and mutant cells under high light. However, a similar quenching effect of formate in the mutant cells is indicative of increased formation of hydroxyl radicals. This observation is further corroborated by higher rate of lipid peroxidation. In addition to this, the superoxide dismutase activity is higher in the mutant (1.2 unit) than in the wild type. Taken together, these results suggest that the cells of the high light-tolerant mutant have an efficient intracellular mechanism to transform the free oxygen radicals.     

PROTECTION OF THE D1 PHOTOSYSTEM II REACTION CENTER PROTEIN FROM DEGRADATION IN ULTRAVIOLET RADIATION FOLLOWING ADAPTATION OF Brassica napus L. TO GROWTH IN ULTRAVIOLET-B

As depletion of the stratospheric ozone layer continues, the biosphere will most likely be exposed to higher levels of ultraviolet-B (UV-B) irradiation (290–320nm). For plants, damage from UV-B can occur at several molecular targets with the photosynthetic apparatus being especially vulnerable. We are interested both in the mechanisms of UV-B-induced damage and identifying adaptation processes that can confer protection from UV-B. Toward this end, Brassica napus (oil seed rape) plants grown under visible light plus a low level of UV-B radiation (adapted plants) were compared to plants grown under visible light alone (control plants). Relative to the control plants, the adapted plants showed little evidence of damage at the levels of morphology or photosynthesis, indicating that B. napus has some tolerance of UV-B and that the plants may have protection mechanisms. Consistent with this, a strong UV-B adaptation process was observed in the plants-accumulation of flavonoids in the epidermis. These pigments seemed to screen a molecular target in the mesophyll. Namely, the D1 photosystem II reaction center protein, which is rapidly degraded in UV-B, was partially protected from degradation in UV-B in the adapted plants. Moreover, the extent that the half-life of the D1 protein increased in the adapted plants was on par with the elevation in total flavonoid concentrations. These experiments demonstrate that degradation of the D1 protein can be used as an in vivo assay of penetration of UV-B photons to the mesophyll.     

Structure and dynamics of photosystem II light-harvesting complex revealed by high-resolution FTICR mass spectrometric proteome analysis

Structure and dynamics of membrane-bound light-harvesting pigment-protein complexes (LHCs), which collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identified and characterized using high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). LHCs from photosystem II (LHCII) were isolated from the thylakoid membrane of Arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel-filtration into LHCII subcomplexes. Using reversed-phase high-performance liquid chromatography and two-dimensional (2D) gel electrophoresis, the LHCII proteins, Lhcb1-6 and fibrillins, were efficiently separated and identified by FTICR-MS. Some of the LHCII subcomplexes were shown to migrate from photosystem II to photosystem I as a result of short-term adaptation to changes in light intensity. In the mobile LHCII subcomplexes, decreased levels of fibrillins and a modified composition of LHCII protein isoforms were identified compared to the tightly bound LHCII subcomplexes. In addition, FTICR-MS analysis revealed several oxidative modifications of LHCII proteins. A number of protein spots in 2D gels were found to contain a mixture of proteins, illustrating the feasibility of high-resolution mass spectrometry to identify proteins that remain unseparated in 2D gels even upon extended pH gradients.     

Dynamics of a myoglobin mutant enzyme: 2D IR vibrational echo experiments and simulations

Myoglobin (Mb) double mutant T67R/S92D displays peroxidase enzymatic activity in contrast to the wild type protein. The CO adduct of T67R/S92D shows two CO absorption bands corresponding to the A(1) and A(3) substates. The equilibrium protein dynamics for the two distinct substates of the Mb double mutant are investigated by using two-dimensional infrared (2D IR) vibrational echo spectroscopy and molecular dynamics (MD) simulations. The time-dependent changes in the 2D IR vibrational echo line shapes for both of the substates are analyzed using the center line slope (CLS) method to obtain the frequency-frequency correlation function (FFCF). The results for the double mutant are compared to those from the wild type Mb. The experimentally determined FFCF is compared to the FFCF obtained from molecular dynamics simulations, thereby testing the capacity of a force field to determine the amplitudes and time scales of protein structural fluctuations on fast time scales. The results provide insights into the nature of the energy landscape around the free energy minimum of the folded protein structure.