Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan (Rapana thomasiana) hemocyanin

Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV–vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60 °C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80 °C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50 kDa functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal β-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90 °C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc.     

Phenoloxidase activity and thermostability of Cancer pagurus and Limulus polyphemus hemocyanin

The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, l-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and l-Dopa (K_m value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for l-Dopa), while k_cat is highest for catechol (2.44 min^? 1 versus 0.67 min^? 1 for l-Dopa and 0.71 min^? 1 for dopamine). On treatment with 4 mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with l-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (T_m ~ 80 °C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (T_m ~ 92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.     

Sex‐linked inheritance of diapause induction in the butterfly Pieris napi

Many temperate insects survive harsh environmental conditions, such as winter, by entering a state of developmental arrest. This diapause state is predominantly induced by photoperiod. The photoperiod varies with latitude and has led to local adaptation in the photoperiodic induction of diapause in many insects. To understand the rapid evolution of the photoperiodic threshold, it is important to investigate and understand the underlying genetic mechanisms. In the present study, the genetic basis of photoperiodic diapause induction is investigated in the green‐veined white butterfly Pieris napi (Lepidoptera, Pieridae) by assaying diapause induction in a range of conditions for a Swedish and Spanish population. Furthermore, the inheritance of diapause induction is assessed in reciprocal F1 hybrids and backcrosses between the two populations. The southern population shows a clear photoperiodic threshold determining diapause or direct development, whereas the northern populations show a high incidence of diapause, regardless of photoperiod. The hybrid crosses reveal that the inheritance of diapause induction is strongly sex‐linked, and that diapause incidence in the genetic crosses is highly dependent on photoperiod. This emphasizes the importance of assaying a range of conditions in diapause inheritance studies. The results indicate a strongly heritable diapause induction with a major component on the Z‐chromosome, as well as a minor effect of the autosomal background.     

Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene

Diesters of phthiocerol and phenolphthiocerol are important virulence factors of Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans. They are both long-chain beta-diols, and their biosynthetic pathway is beginning to be elucidated. Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M. tuberculosis complex examined although phenolphthiocerol diesters are produced by only a few groups of strains. To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M. tuberculosis were analyzed. We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M. tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putative p-hydroxybenzoic acid precursor. We also show that all the strains of M. tuberculosis examined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1 whereas a single open reading frame for pks15/1 is found in Mycobacterium bovis BCG, M. leprae, and strains of M. tuberculosis that produce phenolphthiocerol derivatives. Complementation of the H37Rv strain of M. tuberculosis, which is devoid of phenolphthiocerol derivatives, with the fused pks15/1 gene from M. bovis BCG restored phenolphthiocerol glycolipids production. Conversely, disruption of the pks15/1 gene in M. bovis BCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid. These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to give p-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives.     

AMP-activated protein kinase and coordination of hepatic fatty acid metabolism of starved/carbohydrate-refed rats

Acute increases in the concentration of malonyl-CoA play a pivotal role in mediating the decrease in fatty acid oxidation that occurs in many tissues during refeeding after a fast. In this study, we assess whether such increases in malonyl-CoA in liver could be mediated by malonyl-CoA decarboxylase (MCD), as well as acetyl-CoA carboxylase (ACC). In addition, we examine how changes in the activity of ACC, MCD, and other enzymes that govern fatty acid and glycerolipid synthesis relate temporally to alterations in the activities of the fuel-sensing enzyme AMP-activated protein kinase (AMPK). Rats starved for 48 h and refed a carbohydrate chow diet for 1, 3, 12, and 24 h were studied. Refeeding caused a 40% decrease in the activity of the alpha1-isoform of AMPK within 1 h, with additional decreases in AMPKalpha1 activity and a decrease in AMPKalpha2 occurring between 1 and 24 h. At 1 h, the decrease in AMPK activity was associated with an eightfold increase in the activity of the alpha1-isoform of ACC and a 30% decrease in the activity of MCD, two enzymes thought to be regulated by AMPK. Also, the concentration of malonyl-CoA was increased by 50%. Between 1 and 3 h of refeeding, additional increases in the activity of ACC and decreases in MCD were observed, as was a further twofold increase in malonyl-CoA. Increases in the activity (60%) and abundance (12-fold) of fatty acid synthase occurred predominantly between 3 and 24 h and increases in the activity of mitochondrial sn-glycerol-3-phosphate acyltransferase (GPAT) and acyl-CoA:diaclyglycerol acyltransferase (DGAT) at 12 and 24 h. The results strongly suggest that early changes in the activity of MCD, as well as ACC, contribute to the increase in hepatic malonyl-CoA in the starved-refed rat. They also suggest that the changes in these enzymes, and later occurring increases in enzymes regulating fatty acid and glycerolipid synthesis, could be coordinated by AMPK.     

Mass spectral evidence for N-glycans with branching on fucose in a molluscan hemocyanin

Glycopeptides, isolated from a trypsinolysate of functional unit (FU) RtH2-e of Rapana thomasiana hemocyanin subunit 2, were analysed by electrospray ionization mass spectrometry and MS/MS. From the molecular mass observed after deglycosylation, it was inferred that all glycopeptides shared the same peptide stretch 92-143 of FU RtH2-e with a glycosylation site at Asn-127. Besides the core structure Man(3)GlcNAc(2) for N-glycosylation, structures with a supplementary GlcNAc linked to either the Man(alpha1-3) or the Man(alpha1-6) arm and/or an additional tetrasaccharide unit connected to the other Man arm were observed, indicating the existence of microheterogeneity at the glycan level. The tetrasaccharide unit contains a central fucose moiety substituted with 3-O-methylgalactose and N-acetylgalactosamine, and linked to GlcNAc at the reducing end. This structure represents a novel N-glycan motif and is likely to be immunogenic. A second potential site for N-glycosylation in FU RtH2-e at Asn-17 was shown to be not glycosylated.     

Molecular dissection of the role of two methyltransferases in the biosynthesis of phenolglycolipids and phthiocerol dimycoserosate in the Mycobacterium tuberculosis complex

A few mycobacterial species, most of which are pathogenic for humans, produce dimycocerosates of phthiocerol (DIM) and of glycosylated phenolphthiocerol, also called phenolglycolipid (PGL), two groups of molecules shown to be important virulence factors. The biosynthesis of these molecules is a very complex pathway that involves more than 15 enzymatic steps and has just begun to be elucidated. Most of the genes known to be involved in these pathways are clustered on the chromosome of M. tuberculosis. Based on their amino acid sequences, we hypothesized that the proteins encoded by Rv2952 and Rv2959c, two open reading frames of this locus, are involved in the transfer of methyl groups onto various hydroxyl functions during the biosynthesis of DIM, PGL, and related p-hydroxybenzoic acid derivatives (p-HBAD). Using allelic exchange and site-specific recombination, we produced three recombinant strains of Mycobacterium tuberculosis carrying insertions in Rv2952 or Rv2959c. Analysis of these mutants revealed that (i) the protein encoded by Rv2952 is a methyltransferase catalyzing the transfer of a methyl group onto the lipid moiety of phthiotriol and glycosylated phenolphthiotriol dimycocerosates to form DIM and PGL, respectively, (ii) Rv2959c is part of an operon including the newly characterized Rv2958c gene that encodes a glycosyltransferase also involved in PGL and p-HBAD biosynthesis, and (iii) the enzyme encoded by Rv2959c catalyzes the O-methylation of the hydroxyl group located on carbon 2 of the rhamnosyl residue linked to the phenolic group of PGL and p-HBAD produced by M. tuberculosis. These data further extend our understanding of the biosynthesis of important mycobacterial virulence factors and provide additional tools to decipher the molecular mechanisms of action of these molecules during the pathogenesis of tuberculosis.     

2'-deoxyribonolactone lesion produces G->A transitions in Escherichia coli

2'-deoxyribonolactone (dL) is a C1'-oxidized abasic site damage generated by a radical attack on DNA. Numerous genotoxic agents have been shown to produce dL including UV and gamma-irradiation, ene-dye antibiotics etc. At present the biological consequences of dL present in DNA have been poorly documented, mainly due to the lack of method for introducing the lesion in oligonucleotides. We have recently designed a synthesis of dL which allowed investigation of the mutagenicity of dL in Escherichia coli by using a genetic reversion assay. The lesion was site-specifically incorporated in a double-stranded bacteriophage vector M13G*1, which detects single-base-pair substitutions at position 141 of the lacZalpha gene by a change in plaque color. In E.coli JM105 the dL-induced reversion frequency was 4.7 x 10(-5), similar to that of the classic abasic site 2'-deoxyribose (dR). Here we report that a dL residue in a duplex DNA codes mainly for thymidine. The processing of dL in vivo was investigated by measuring lesion-induced mutation frequencies in DNA repair deficient E.coli strains. We showed a 32-fold increase in dL-induced reversion rate in AP endonuclease deficient (xth nfo) mutant compared with wild-type strain, indicating that the Xth and Nfo AP endonucleases participate in dL repair in vivo.