Exploring the molecular mechanisms that underpin key biological processes, the Biochemical Journal is a leading bioscience journal publishing high-impact scientific research papers and reviews on the latest advances and new mechanistic concepts in the fields of biochemistry, cellular biosciences and molecular biology. The Journal and its Editorial Board are committed to publishing work that provides a significant advance to current understanding or mechanistic insights; studies that go beyond observational work using in vitro and/or in vivo approaches are welcomed. Painless publishing: All papers undergo a rigorous peer review process; however, the Editorial Board is committed to ensuring that, if revisions are recommended, extra experiments not necessary to the paper will not be asked for. Areas covered in the journal include: Cell biology Chemical biology Energy processes Gene expression and regulation Mechanisms of disease Metabolism Molecular structure and function Plant biology Signalling
Cristina I. Caescu, Grace R. Jeschke, Benjamin E. Turk
The metalloproteinases TACE [tumour necrosis factor α-converting enzyme; also known as ADAM17 (a disintegrin and metalloproteinase 17)] and ADAM10 are the primary enzymes responsible for catalysing release of membrane-anchored proteins from the cell surface in metazoan organisms. Although the repertoire of protein substrates for these two proteases is partially overlapping, each one appears to target a subset of unique proteins in vivo. The mechanisms by which the two proteases achieve specificity for particular substrates are not completely understood. We have used peptide libraries to define the cleavage site selectivity of TACE and ADAM10. The two proteases have distinct primary sequence requirements at multiple positions surrounding the cleavage site in their substrates, which allowed us to generate peptide substrates that are highly specific for each of these proteases. The major difference between the two protease specificities maps to the P1′ position (immediately downstream of the cleavage site) of the substrate. At this position, TACE is selective for smaller aliphatic residues, whereas ADAM10 can accommodate aromatic amino acids. Using mutagenesis we identified three residues in the S1′ pockets of these enzymes that dramatically influence specificity for both peptide and protein substrates. Our results suggest that substrate selectivity of TACE and ADAM10 can be at least partly rationalized by specific features of their active sites.
The major excreted protein (MEP) purified from Kirsten-virus-transformed 3T3 fibroblasts and mature human cathepsin L were compared in respect to a number of catalytic criteria and found to be similar. The Mr of MEP is 39,000, whereas that of mature human cathepsin L is 30,000. Sequence data suggested that MEP could be a pro-form of mouse cathepsin L. Both enzymes acted on the synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide with similar catalytic constants and acted optimally at pH 5.5. Both were rapidly inactivated by the active-site-directed inhibitors benzyloxycarbonyl-Phe-Phe-diazomethane and L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane, and furthermore, 3H-labelled L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-acetamid o)butane, which binds covalently to the heavy chain of mature cathepsin L, also bound to MEP. MEP autolyses rapidly at pH 3.0 to give lower-Mr (35,000 and 30,000) forms, but all forms react with the radiolabelled inhibitor. No autolysis occurred above pH 5.0. MEP hydrolysed azocasein at pH 5.0, demonstrating that it is capable of hydrolysing protein substrates without autolytic activation. Unlike mature forms of cathepsin L, MEP is stable, but not active, at neutral pH. The present work shows that cathepsin L can be secreted as a higher-Mr precursor that is stable in extracellular fluids but only active where local pH values fall below 6.0. These results suggest that the extra N-terminal peptide on MEP is not an activation peptide, but is a regulatory peptide affecting the pH-stability and activity of mouse cathepsin L.
Alan E. Senior, J. Allan Downie, G B Cox, F. Gibson, L Langman, D R Fayle
Four mutant strains of Escherichia coli which lack membrane-bound adenosine triphosphatase activity were shown by genetic-complementation tests to carry mutations in the uncA gene. A soluble inactive F1-ATPase aggregate was released from the membranes of three of the uncA mutant strains by low-ionic-strength washing, and purified by procedures developed for the purification of F1-ATPase from normal strains. Analysis of the subunit structure by two-dimensional gel electrophoresis indicated that the F1-ATPase in strains carrying the uncA401 or uncA453 alleles had a subunit structure indistinguishable from normal F1-ATPase. In contrast, the F1-ATPase from the strain carrying the uncA447 allele contained an alpha-subunit of normal molecular weight, but abnormal net charge. Membranes from strains carrying the uncA450 allele did not have F1-ATPase aggregates that could be solubilized by low-ionic-strength washing. However, a partial dipolid strain carrying both the uncA+ and uncA450 alleles formed an active F1-ATPase aggregate which could be solubilized by low-ionic-strength washing of the membranes and which contained two types of alpha-subunit, one of which was normal and the other had abnormal net charge. It is concluded that the uncA gene codes for the alpha-subunit of the adenosine triphosphatase.
John G. Wise, T. Michael Duncan, Lisa R. Latchney, Daniel N. Cox, Alan E. Senior
Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5′-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5′-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for ‘promotion’ of catalysis in this enzyme.
Cell membranes from etiolated Pisum sativum (pea) tissues were separated by ultracentrifugation on linear sucrose density gradients and assayed for membrane marker and glycosyltransferase activity. Membrane fractions were shown to incorporate glucose from UDP-D-[14C]glucose into polysaccharides with glycosyl linkages consistent with synthesis of xyloglucan. A combined assay using g.c., radiogas proportional counting and m.s. was employed to determine the identities of 14C-labelled glycosyl residues and the glycosyl linkages between them. In glucan synthase I assays, membrane fractions enriched for Golgi membranes showed 14C incorporation into 4- and 4,6-glucose residues, with minor incorporation into 3-glucose residues. In glucan synthase II assays, all 14C incorporation was into 3- and 3,4-glucose. There was a shift in glycosyl linkage of 14C incorporation from predominantly 4-glucose at low UDP-glucose concentration to predominantly 3- and 3,4-glucose at high UDP-glucose concentrations. Mn2+ stimulated incorporation of radioactivity into 4,6-glucose residues characteristic of xyloglucan polysaccharides. Addition of exogenous UDP-xylose to assay mixtures stimulated incorporation into 4,6-glucose, with a maximum at 15 microM UDP-xylose.
A method was devised to purify branched-chain oxo acid dehydrogenase (BCOAD) from rat kidney which retains endogenous kinase activity. Incorporation of 32P into purified enzyme parallels the time course of enzyme inhibition by ATP. Phosphorylation occurs on a serine residue(s) of the 46000-mol.wt. subunit of the enzyme complex. Endogenous phosphatase activity is not present after purification, and added pyruvate dehydrogenase phosphate phosphatase does not re-activate BCOAD or liberate 32P from previously labelled enzyme. These results demonstrate that BCOAD can be regulated by an endogenous protein kinase and that the phosphorylation-cycle enzymes regulating BCOAD appear to be distinct from those associated with pyruvate dehydrogenase complex.
David Carling, Claire Thornton, Angela Woods, Matthew J. Sanders
The hydrolysis of ATP drives virtually all of the energy-requiring processes in living cells. A prerequisite of living cells is that the concentration of ATP needs to be maintained at sufficiently high levels to sustain essential cellular functions. In eukaryotic cells, the AMPK (AMP-activated protein kinase) cascade is one of the systems that have evolved to ensure that energy homoeostasis is maintained. AMPK is activated in response to a fall in ATP, and recent studies have suggested that ADP plays an important role in regulating AMPK. Once activated, AMPK phosphorylates a broad range of downstream targets, resulting in the overall effect of increasing ATP-producing pathways whilst decreasing ATP-utilizing pathways. Disturbances in energy homoeostasis underlie a number of disease states in humans, e.g. Type 2 diabetes, obesity and cancer. Reflecting its key role in energy metabolism, AMPK has emerged as a potential therapeutic target. In the present review we examine the recent progress aimed at understanding the regulation of AMPK and discuss some of the latest developments that have emerged in key areas of human physiology where AMPK is thought to play an important role.
Sarah E. Dixon-Clarke, Saifeldin N. Shehata, T. Krojer, Timothy Sharpe, F. von Delft, Kei Sakamoto, Alex N. Bullock
CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts.
Sarah Traverse, Néstor Gómez, Hugh Paterson, C. J. Marshall, Philip Cohen
Stimulation of PC12 cells with nerve growth factor (NGF) increased mitogen-activated protein kinase kinase (MAPKK) activity > 20-fold after 5 min to a level that was largely sustained for at least 90 min. MAPKK activity was stimulated to a similar level by epidermal growth factor (EGF), but peaked at 2 min, declining thereafter and returning to basal levels after 60-90 min. Activation of MAPKK by either growth factor occurred prior to the activation of MAP kinase, consistent with MAPKK being the physiological activator of MAP kinase. The results demonstrate that the transient activation of MAPKK by EGF and its sustained activation by NGF underlies the transient and sustained activation of MAP kinase induced by EGF and NGF respectively. NGF or EGF induced the same two forms of MAPKK that were resolved on a Mono Q column. The Peak-1 MAPKK was activated initially and partially converted into the more acidic peak-2 MAPKK after prolonged growth-factor stimulation. The Peak-2 MAPKK was 20-fold more sensitive to inactivation by the catalytic subunit of protein phosphatase 2A. Stimulation with NGF caused a striking translocation of MAP kinase from the cytosol to the nucleus after 30 min, but not nuclear translocation of MAP kinase occurred after stimulation with EGF. The results suggest that sustained activation of the MAP kinase cascade may be required for MAP kinase to enter the nucleus, where it may initiate the gene transcription events required for neuronal differentiation of PC12 cells.
Sergio Papa, F. Guerrieri, Michele Lorusso, G. Izzo, Domenico Boffoli, Ferdinando Capuano, Nazzareno Capitanio, Nicola Altamura
1. The →H+/e− quotients for proton release from mitochondria associated with electron flow from succinate and duroquinol to O2, ferricyanide or ferricytochrome c, and from NNN′N′-tetramethyl-p-phenylenediamine+ascorbate to O2, were determined from rate measurements of electron flow and proton translocation. 2. Care was taken to avoid, or to take into account, unrelated electron flow and proton translocation, which might take place in addition to the oxido-reductions that were the subject of our analysis. Spectrophotometric techniques were chosen to provide accurate measurement of the rate of consumption of oxidants and reductants. The rate of proton translocation was measured with fast pH meters with a precision of 10−3 pH unit. 3. The →H+/O quotient for succinate or duroquinol oxidation was, at neutral pH, 4, when computed on the basis of spectrophotometric determinations of the rate of O2 consumption or duroquinol oxidation. Higher →H+/O quotients for succinate oxidation, obtained from polarographic measurements of O2 consumption, resulted from underestimation of the respiratory rate. 4. The →H+/2e− quotient for electron flow from succinate and duroquinol to ferricyanide or ferricytochrome c ranged from 3.9 to 3.6. 5. Respiration elicited by NNN′N′-tetramethyl-p-phenylenediamine+ascorbate by antimycin-inhibited mitochondria resulted in extra proton release in addition to that produced for oxidation of ascorbate to dehydroascorbate. Accurate spectrophotometric measurement of respiration showed that the →H+/e− ratio was only 0.25 and not 0.7–1.0 as obtained with the inadequate polarographic assay of respiration. Proton release was practically suppressed when mitochondria were preincubated aerobically in the absence of antimycin. Furthermore, the rate of scalar proton consumption for water production was lower than that expected from the stoicheiometry. Thus the extra proton release observed during respiration elicited by NNN′N′-tetramethyl-p-phenylenediamine+ascorbate is caused by oxidation of endogenous hydrogenated reductants. 6. It is concluded that (i) the →H+/O quotient for the cytochrome system is, at neutral pH, 4 and not 6 or 8 as reported by others; (ii) all the four protons are released during electron flow from quinol to cytochrome c; (iii) the oxidase transfers electrons from cytochrome c to protons from the matrix aqueous phase and does not pump protons from the matrix to the outer aqueous phase.
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