A hexokinase effect in the inhibition of oxidative phosphorylation in heart muscle mitochondria by Adriamycin

The inhibitory action of the anticancer antibiotic, Adriamycin, on succinate-dependent oxidative phosphorylation in heart mitochondria was markedly potentiated by the presence of hexokinase in the reaction medium. This 'hexokinase effect' was not observed in the oxidation of NAD+-linked substrates, or when liver or kidney mitochondria were used in place of heart mitochondria. These results offer a biochemical explanation for the extreme cardiac toxicity of the drug.

Inhibition of mitochondrial oxidative phosphorylation by 2-methyl-4-dimethylaminoazobenzene

The azodye 2-methyl-4-dimethylaminoazobenzene inhibited oxidation and phosphorylation in tightly coupled rat liver mitochondria. Phosphorylation was more sensitive to the inhibitory action of the azodye than was the oxidation of succinate or ascorbate. The oxidation of NAD+-linked substrate was severely inhibited by the compound. In submitochondrial particles, only NADH oxidation was sensitive. The site of inhibition has been identified to lie between the dehydrogenase flavoprotein and ubiquinone.

Alamethicin and synthetic peptide fragments as uncouplers of mitochondrial oxidative phosphorylation. Effect of chain length and change

Alamethicin, its derivatives and some synthetic fragments have been shown to be uncouplers of oxidative phosphorylation in rat liver mitochondria. A minimum peptide chain length of 13 residues is necessary for this activity. Peptide esters are more efficient uncouplers than the corresponding peptide acids. Esterification of the Glu(18) γ-COOH group in alamethicin does not diminish uncoupling activity. The structural requirements for uncoupling activity parallel those determined for ionophoretic action in small, unilamellar liposomes. Aib, α-aminoisobutyric acid; Z, benzyloxycarbonyl; OMe, methyl ester; OBz, benzyl ester; Ac, acetyl; CTC, chlortetracycline.

Movement-associated proteins of Potato virus A: attachment to virus particles and phosphorylation

The particles of Potato virus A (PVA; genus Potyvirus) are helically constructed filaments that contain multiple copies of a single type of coat-protein (CP) subunit and a single copy of genome-linked protein (VPg), attached to one end of the virion. Examination of negatively-stained virions by electron microscopy revealed flexuous, rod-shaped particles with no obvious terminal structures. It is known that particles of several filamentous plant viruses incorporate additional minor protein components, forming stable complexes that mediate particle disassembly, movement or transmission by insect vectors. The first objective of this work was to study the interaction of PVA movement-associated proteins with virus particles and how these interactions contribute to the morphology and function of the virus particles. Purified particles of PVA were examined by atomic force microscopy (AFM) and immuno-gold electron microscopy. A protrusion was found at one end of some of the potyvirus particles, associated with the 5' end of the viral RNA. The tip contained two virus-encoded proteins, the genome-linked protein (VPg) and the helper-component proteinase (HC-Pro). Both are required for cell-to-cell movement of the virus. Biochemical and electron microscopy studies of purified PVA samples also revealed the presence of another protein required for cell-to-cell movement the cylindrical inclusion protein (CI), which is also an RNA helicase/ATPase. Centrifugation through a 5-40% sucrose gradient separated virus particles with no detectable CI to a fraction that remained in the gradient, from the CI-associated particles that went to the pellet. Both types of particles were infectious. AFM and translation experiments demonstrated that when the viral CI was not present in the sample, PVA virions had a beads-on-a-string phenotype, and RNA within the virus particles was more accessible to translation. The second objective of this work was to study phosphorylation of PVA movement-associated and structural proteins (CP and VPg) in vitro and, if possible, in vivo. PVA virion structural protein CP is necessary for virus cell-to-cell movement. The tobacco protein kinase CK2 was identified as a kinase phosphorylating PVA CP. A major site of CK2 phosphorylation in PVA CP was identified as a single threonine within a CK2 consensus sequence. Amino acid substitutions affecting the CK2 consensus sequence in CP resulted in viruses that were defective in cell-to-cell and long-distance movement. The CK2 regulation of virion assembly and cell-to-cell movement by phosphorylation of CP was possibly due to the inhibition of CP binding to viral RNA. Four putative phosphorylation sites were identified from an in vitro phosphorylated recombinant VPg. All four were mutated and the spread of mutant viruses in two different host plants was studied. Two putative phosphorylation site mutants (Thr45 and Thr49) had phenotypes identical to that of a wild type (WT) virus infection in both Nicotiana benthamiana and N. tabacum plants. The other two mutant viruses (Thr132/Ser133 and Thr168) showed different phenotypes with increased or decreased accumulation rates, respectively, in inoculated and the first two systemically infected leaves of N. benthamiana. The same mutants were occasionally restricted to single cells in N. tabacum plants, suggesting the importance of these amino acids in the PVA infection cycle in N. tabacum.

Cooperativity in the inhibition of oxidative phosphorylation by chlorophenoxyisobutyrate

The kinetics of inhibition of oxidative phosphorylation by the antihypercholes-terolaemic compound p-chlorophenoxyisobutyrate reveal cooperativity characteristic of allosteric interactions. Hill plots and Dixon plots give clear indication that the compound interferes with two distinct steps in the energy-transfer pathway. The values of interaction coefficients calculated from the Hill plots were two and four in the direction of ATP synthesis and one and two in the reverse direction. This could mean either that the pathways of synthesis and breakdown of ATP are different, or that if the pathways are the same, only half the inhibitor-binding sites function in the reverse direction.

LFA-1 Integrin beta2 Chain Phosphorylation Regulates Protein Interactions and Mediates Signals in T Cells

Integrins are heterodimeric transmembrane adhesion receptors composed of alpha- and beta-subunits and they are vital for the function of multicellular organisms. Integrin-mediated adhesion is a complex process involving both affinity regulation and coupling to the actin cytoskeleton. Integrins also function as bidirectional signaling devices, regulating cell adhesion and migration after inside-out signaling, but also signal into the cell to regulate growth, differentiation and apoptosis after ligand binding. The LFA-1 integrin is exclusively expressed in leukocytes and is of fundamental importance for the function of the immune system. The LFA-1 integrins have short intracellular tails, which are devoid of catalytic activity. These cytoplasmic domains are important for integrin regulation and both the alpha and beta chain become phosphorylated. The alpha chain is constitutively phosphorylated, but the beta chain becomes phosphorylated on serine and functionally important threonine residues only after cell activation. The cytoplasmic tails of LFA-1 bind to many cytoskeletal and signaling proteins regulating numerous cell functions. However, the molecular mechanisms behind these interactions have been poorly understood. Phosphorylation of the cytoplasmic tails of the LFA-1 integrin could provide a mechanism to regulate integrin-mediated cytoskeletal interactions and take part in T cell signaling. In this study, the effects of phosphorylation of LFA-1 integrin cytoplasmic tails on different cellular functions were examined. Site-specific phosphorylation of both the alpha- and beta-chains of the LFA-1 was shown to have a role in the regulation of the LFA-1 integrin.Alpha-chain Ser1140 is needed for integrin conformational changes after chemokine- or integrin ligand-induced activation or after activation induced by active Rap1, whereas beta-chain binds to 14-3-3 proteins through the phosphorylated Thr758 and mediates cytoskeletal reorganization. Thr758 phosphorylation also acts as a molecular switch to inhibit filamin binding and allows 14-3-3 protein binding to integrin cytoplasmic domain, and it was also shown to lead to T cell adhesion, Rac-1/Cdc42 activation and expression of the T cell activation marker CD69, indicating a signaling function for Thr758 phosphorylation in T cells. Thus, phosphorylation of the cytoplasmic tails of LFA-1 plays an important role in different functions of the LFA-1 integrin in T cells. It is of vital importance to study the mechanisms and components of integrin regulation since leukocyte adhesion is involved in many functions of the immune system and defects in the regulation of LFA-1 contributes to auto-immune diseases and fundamental defects in the immune system.

A phosphorylation-induced turn defines the Alzheimer's disease AT8 antibody epitope on the tau protein

Post mortem biochemical staging of Alzheimer’s disease is currently based on immunochemical analysis of brain slices with the AT8 antibody. The epitope of AT8 is described around the pSer202/pThr205 region of the hyperphosphorylated form of the neuronal protein tau. In this study, NMR spectroscopy was used to precisely map the AT8 epitope on phosphorylated tau, and derive its defining structural features by a combination of NMR analyses and molecular dynamics. A particular turn conformation is stabilized by a hydrogen bond of the phosphorylated Thr205 residue to the amide proton of Gly207, and is further stabilized by the two Arg residues opposing the pSer202/pThr205.

Molecular dynamics simulation of the phosphorylation-induced conformational changes of a tau peptide fragment

Aggregation of the microtubule associated protein tau (MAPT) within neurons of the brain is the leading cause of tauopathies such as Alzheimer's disease. MAPT is a phospho-protein that is selectively phosphorylated by a number of kinases in vivo to perform its biological function. However, it may become pathogenically hyperphosphorylated, causing aggregation into paired helical filaments and neurofibrillary tangles. The phosphorylation induced conformational change on a peptide of MAPT (htau225−250) was investigated by performing molecular dynamics simulations with different phosphorylation patterns of the peptide (pThr231 and/or pSer235) in different simulation conditions to determine the effect of ionic strength and phosphate charge. All phosphorylation patterns were found to disrupt a nascent terminal β-sheet pattern (226VAVVR230 and 244QTAPVP249), replacing it with a range of structures. The double pThr231/pSer235 phosphorylation pattern at experimental ionic strength resulted in the best agreement with NMR structural characterization, with the observation of a transient α-helix (239AKSRLQT245). PPII helical conformations were only found sporadically throughout the simulations. Proteins 2014; 82:1907–1923. © 2014 Wiley Periodicals, Inc.

Identification of IDUA and WNT16 phosphorylation-related non-synonymous polymorphisms for bone mineral density in meta-analyses of genome-wide association studies

Protein phosphorylation regulates a wide variety of cellular processes. Thus, we hypothesize that single-nucleotide polymorphisms (SNPs) that may modulate protein phosphorylation could affect osteoporosis risk. Based on a previous conventional genome-wide association (GWA) study, we conducted a three-stage meta-analysis targeting phosphorylation-related SNPs (phosSNPs) for femoral neck (FN)-bone mineral density (BMD), total hip (HIP)-BMD, and lumbar spine (LS)-BMD phenotypes. In stage 1, 9593 phosSNPs were meta-analyzed in 11,140 individuals of various ancestries. Genome-wide significance (GWS) and suggestive significance were defined by α = 5.21 × 10–6 (0.05/9593) and 1.00 × 10–4, respectively. In stage 2, nine stage 1–discovered phosSNPs (based on α = 1.00 × 10–4) were in silico meta-analyzed in Dutch, Korean, and Australian cohorts. In stage 3, four phosSNPs that replicated in stage 2 (based on α = 5.56 × 10–3, 0.05/9) were de novo genotyped in two independent cohorts. IDUA rs3755955 and rs6831280, and WNT16 rs2707466 were associated with BMD phenotypes in each respective stage, and in three stages combined, achieving GWS for both FN-BMD (p = 8.36 × 10–10, p = 5.26 × 10–10, and p = 3.01 × 10–10, respectively) and HIP-BMD (p = 3.26 × 10–6, p = 1.97 × 10–6, and p = 1.63 × 10–12, respectively). Although in vitro studies demonstrated no differences in expressions of wild-type and mutant forms of IDUA and WNT16B proteins, in silico analyses predicts that WNT16 rs2707466 directly abolishes a phosphorylation site, which could cause a deleterious effect on WNT16 protein, and that IDUA phosSNPs rs3755955 and rs6831280 could exert indirect effects on nearby phosphorylation sites. Further studies will be required to determine the detailed and specific molecular effects of these BMD-associated non-synonymous variants. © 2015 American Society for Bone and Mineral Research.

Structural Modes of Stabilization of Permissive Phosphorylation Sites in Protein Kinases: Distinct Strategies in Ser/Thr and Tyr Kinases

Protein kinases phosphorylate several cellular proteins providing control mechanisms for various signalling processes. Their activity is impeded in a number of ways and restored by alteration in their structural properties leading to a catalytically active state. Most protein kinases are subjected to positive and negative regulation by phosphorylation of Ser/Thr/Tyr residues at specific sites within and outside the catalytic core. The current review describes the analysis on 3D structures of protein kinases that revealed features distinct to active states of Ser/Thr and Tyr kinases. The nature and extent of interactions among well-conserved residues surrounding the permissive phosphorylation sites differ among the two classes of enzymes. The network of interactions of highly conserved Arg preceding the catalytic base that mediates stabilization of the activation segment exemplifies such diverse interactions in the two groups of kinases. The N-terminal and the C-terminal lobes of various groups of protein kinases further show variations in their extent of coupling as suggested from the extent of interactions between key functional residues in activation segment and the N-terminal αC-helix. We observe higher similarity in the conformations of ATP bound to active forms of protein kinases compared to ATP conformations in the inactive forms of kinases. The extent of structural variations accompanying phosphorylation of protein kinases is widely varied. The comparison of their crystal structures and the distinct features observed are hoped to aid in the understanding of mechanisms underlying the control of the catalytic activity of distinct subgroups of protein kinases.

Involvement of Synthesis and Phosphorylation of Nuclear Protein Factors That Bind to the Positivecis-Acting Element in the Transcriptional Activation of the CYP2B1/B2 Gene by Phenobarbitonein Vivo

The synthesis and phosphorylation of protein factor(s) that bind to the positivecis-acting element (−69 to −98 nt) of the CYP2B1/B2 gene have been examinedin vivoin the rat. Treatment of rats with cycloheximide, a protein synthetic inhibitor, suppresses basal as well as phenobarbitone-induced levels of CYP2B1/B2 mRNA and its run-on transcription. Under these conditions, complex formation of the nuclear extract with the positive element is also inhibited, as judged by gel shift assays. Treatment of rats with 2-aminopurine, a general protein kinase inhibitor, blocks the phenobarbitone-mediated increase in CYP2B1/B2 mRNA, cell-free transcription of a minigene construct containing the positive element, pP450e179DNA, and binding of nuclear proteins to the positive element. Treatment of rats with okadaic acid, a protein phosphatase inhibitor, mimics the effects of phenobarbitone, but only partially. Thus, both phenobarbitone and okadaic acid individually enhance binding of the nuclear protein(s) to the positive element, cell-free transcription of the minigene construct, and phosphorylation of the not, vert, similar26- and 94-kDa proteins binding to the positive element. But unlike phenobarbitone, okadaic acid is not an inducer of CYP2B1/B2 mRNA or its run-on transcription. Thus, phenobarbitone-responsive positive element interactions constitute only a minimal requirement, and okadaic acid is perhaps not able to bring about the total requirement for activation of CYP2B1/B2 gene transcription that should include interaction between the minimal promoter and further upstream elements. An intriguing feature is the antagonistic effect of okadaic acid on phenobarbitone-mediated effects on CYP2B1/B2 mRNA levels, cell-free and run-on transcription, and nuclear protein binding to the positive element. The reason for this antagonism is not clear. It is concluded that phenobarbitone treatment enhancesin vivothe synthesis and phosphorylation of protein factors binding to the positive element and these constitute a minimal requirement for the transcriptional activation of the CYP2B1/B2 gene.

Inhibition of Tyrosine Phosphorylation of Sperm Flagellar Proteins, Outer Dense Fiber Protein-2 and Tektin-2, Is Associated With Impaired Motility During Capacitation of Hamster Spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Phosphorylation status of the phosphoprotein P of rinderpest virus modulates transcription and replication of the genome

The phosphoprotein P of paramyxoviruses is known to play more than one role in genome transcription and replication. Phosphorylation of P at the NH2 terminus by cellular casein kinase II has been shown to be necessary for transcription of the genome in some of the viruses, while it is dispensable for replication. The phosphorylation null mutant of rinderpest virus P protein, in which three serine residues have been mutated, has been shown earlier to be non-functional in an in vivo minigenome replication/transcription system. In this work, we have shown that the phosphorylation of P protein is essential for transcription, whereas the null mutant is active in replication of the genome in vivo. The null mutant P acts as a transdominant repressor of transcriptional activity of wild-type P and as an activator of replication carried out by wild-type P protein. These results suggest the phosphorylation status of P may act as a replication switch during virus replication. We also show that the phosphorylation null mutant P is capable of interacting with L and N proteins and is able to form a tripartite complex of L-(N-P) when expressed in insect cells, similar to wild-type P protein.