Active Enzymatic Complex Research Articles

Targetable leukaemia dependency on noncanonical PI3Kγ signalling.

Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.

Dual Role of ACBD6 in the Acylation Remodeling of Lipids and Proteins.

The transfer of acyl chains to proteins and lipids from acyl-CoA donor molecules is achieved by the actions of diverse enzymes and proteins, including the acyl-CoA binding domain-containing protein ACBD6. N-myristoyl-transferase (NMT) enzymes catalyze the covalent attachment of a 14-carbon acyl chain from the relatively rare myristoyl-CoA to the N-terminal glycine residue of myr-proteins. The interaction of the ankyrin-repeat domain of ACBD6 with NMT produces an active enzymatic complex for the use of myristoyl-CoA protected from competitive inhibition by acyl donor competitors. The absence of the ACBD6/NMT complex in ACBD6.KO cells increased the sensitivity of the cells to competitors and significantly reduced myristoylation of proteins. Protein palmitoylation was not altered in those cells. The specific defect in myristoyl-transferase activity of the ACBD6.KO cells provided further evidence of the essential functional role of the interaction of ACBD6 with the NMT enzymes. Acyl-CoAs bound to the acyl-CoA binding domain of ACBD6 are acyl donors for the lysophospholipid acyl-transferase enzymes (LPLAT), which acylate single acyl-chain lipids, such as the bioactive molecules LPA and LPC. Whereas the formation of acyl-CoAs was not altered in ACBD6.KO cells, lipid acylation processes were significantly reduced. The defect in PC formation from LPC by the LPCAT enzymes resulted in reduced lipid droplets content. The diversity of the processes affected by ACBD6 highlight its dual function as a carrier and a regulator of acyl-CoA dependent reactions. The unique role of ACBD6 represents an essential common feature of (acyl-CoA)-dependent modification pathways controlling the lipid and protein composition of human cell membranes.

The Dual Oxidase Duox2 stabilized with DuoxA2 in an enzymatic complex at the surface of the cell produces extracellular H2O2 able to induce DNA damage in an inducible cellular model

Thyroid hormone synthesis requires H2O2, produced by two NADPH oxidases, Duox1 and Duox2. To be fully active at the apical pole of the thyrocytes, these enzymes need additional maturation factors DuoxA1 and DuoxA2. The proteins have been shown to be localized at the cell surface, suggesting that they could form a complex with Duox counterparts. We have generated multiple HEK293 Tet-On3G cell lines that express various combinations of DuoxA upon doxycycline induction, in association with a constitutive expression of the Duox enzyme. We compared Duox specific activity, Duox/DuoxA cell surface interactions and the cellular consequences of sustained H2O2 generation. By normalizing H2O2 extracellular production by Duox or DuoxA membrane expression, we have demonstrated that the most active enzymatic complex is Duox2/DuoxA2, compared to Duox1/DuoxA1. A direct cell surface interaction was shown between Duox1/2 and both DuoxA1 and DuoxA2 using the Duolink® technology, Duox1/DuoxA1 and Duox2/DuoxA2 membrane complexes being more stable than the unpaired ones. A significant increase in DNA damage was observed in the nuclei of Duox2/DuoxA2 expressing cells after doxycycline induction and stimulation of Duox catalytic activity. The maturation and activity of Duox2 were drastically impaired when expressed with the glycosylation-defective maturation factor DuoxA2, while the impact of the unglycosylated DuoxA1 mutant on Duox1 membrane expression and activity was rather limited. The present data demonstrate for the first time that H2O2 produced by the Duox2/DuoxA2 cell surface enzymatic complex could provoke potential mutagenic DNA damage in an inducible cellular model, and highlight the importance of the co-expressed partner in the activity and stability of Duox/DuoxA complexes.

Are Cellulosome Scaffolding Protein CipC and CBM3-Containing Protein HycP, Involved in Adherence of Clostridium cellulolyticum to Cellulose?

Clostridium cellulolyticum, a mesophilic anaerobic bacterium, produces highly active enzymatic complexes called cellulosomes. This strain was already shown to bind to cellulose, however the molecular mechanism(s) involved is not known. In this context we focused on the gene named hycP, encoding a 250-kDa protein of unknown function, containing a Family-3 Carbohydrate Binding Module (CBM3) along with 23 hyaline repeat modules (HYR modules). In the microbial kingdom the gene hycP is only found in C. cellulolyticum and the very close strain recently sequenced Clostridium sp BNL1100. Its presence in C. cellulolyticum guided us to analyze its function and its putative role in adhesion of the cells to cellulose. The CBM3 of HycP was shown to bind to crystalline cellulose and was assigned to the CBM3b subfamily. No hydrolytic activity on cellulose was found with a mini-protein displaying representative domains of HycP. A C. cellulolyticum inactivated hycP mutant strain was constructed, and we found that HycP is neither involved in binding of the cells to cellulose nor that the protein has an obvious role in cell growth on cellulose. We also characterized the role of the cellulosome scaffolding protein CipC in adhesion of C. cellulolyticum to cellulose, since cellulosome scaffolding protein has been proposed to mediate binding of other cellulolytic bacteria to cellulose. A second mutant was constructed, where cipC was inactivated. We unexpectedly found that CipC is only partly involved in binding of C. cellulolyticum to cellulose. Other mechanisms for cellulose adhesion may therefore exist in C. cellulolyticum. In addition, no cellulosomal protuberances were observed at the cellular surface of C. cellulolyticum, what is in contrast to reports from several other cellulosomes producing strains. These findings may suggest that C. cellulolyticum has no dedicated molecular mechanism to aggregate the cellulosomes at the cellular surface.

Elevation of free proline and proline-rich protein levels by simultaneous manipulations of proline biosynthesis and degradation in plants

Proline-rich proteins (PRP) are cell wall and plasma membrane-anchored factors involved in cell wall maintenance and its stress-induced fortification. Here we compare the synthesis of P5C as the proline (Pro) precursor in the cytosol and chloroplast by an introduced alien system and evaluate correlation between PRP synthesis and free Pro accumulation in plants. We developed a Pro over-producing system by generating transgenic tobacco plants overexpressing E. coli P5C biosynthetic enzymes; Pro-indifferent gamma-glutamyl kinase 74 (GK74) and gamma-glutamylphosphate reductase (GPR), as well as antisensing proline dehydrogenase ( ProDH) transcription. GK74 and GPR enzymes were targeted either to the cytosol or plastids. Molecular analyses indicated that the two bacterial enzymes are efficiently expressed in plant cells, correctly targeted to the cytosol or chloroplasts, and processed to active enzymatic complexes in the two compartments. Maximal Pro increase is obtained when GK74 and GPR are active in chloroplasts, and ProDH mRNA level is reduced by anti-sense silencing, resulting in more than 50-fold higher Pro content compared to that of wild type tobacco plants. The Pro over-producing system efficiently works in tobacco and Arabidopsis. The elevation of Pro levels promotes accumulation of ectopically expressed Cell Wall Linker Protein (AtCWLP), a membrane protein with an external Pro-rich domain. These results suggest that the Pro-generating system can support endogenous or alien PRP production in plants.

Effect of Biological and Biochemical Silage Additives on Final Nutritive, Hygienic and Fermentation Characteristics of Ensiled High Moisture Crimped Corn

The aim of this study was to determine the effect of different biological and biochemical additives on the final nutritive quality, fermentation process and concentration of mycotoxins of ensiled high moisture crimped corn. We created four variants for the experiment: control (UC), A1, and A2 (biological stimulators with the active principle of lactic acid bacteria) and variant B (combined additives with the active principle of lactic acid bacteria, benzoate sodium and active enzymatic complex of cellulases). After 6 months of storage in laboratory conditions, we determined in experimental silages the content of dry matter ranging from 608.9 to 613 g kg-1. The significantly lower content of crude fibre was detected in silages with additives. In silages ensiled with additives we detected the highest content of nitrogen-free extract in variant B (834.3 g kg-1 of DM, P < 0.05). A similar effect was determined also in the content of starch; significant differences were detected in variants A1 and B (P < 0.05) compared to the control variant. We detected a significantly (P < 0.05) higher content of total sugars in trial silages; the highest content was in variant A2 (6.1 g kg-1 of DM). In the trial variants we determined significantly the lowest content of acetic acid in variant B (2.82 g kg-1 of DM). In case of butyric acid, whose content in the control variant was 0.22 g kg-1 of DM, we detected the lowest content in variant A1 conserved with homo- and heterofermentative species of lactic acid bacteria. The lowest content of ammonia was determined in silages of variant B (0.074 g kg-1 of DM). We found lower concentrations of DON and FUM (P > 0.05) after the application of biological and biochemical silage additives. In concentration of T-2 toxin we detected a significantly (P < 0.05) lower value in variant A1. In concentration of AFL we found significant differences between variants A1 and B, as well as in concentration of OT between untreated control variant (UC) and variants conserved by additives. Application of silage additives influenced the nutritive and hygienic quality of the conserved fodders.

Succinate dehydrogenase gene arrangement and expression in Anaplasma phagocytophilum

DNA sequencing of the region directly downstream of the Anaplasma phagocytophilum (strain MRK) 16S rRNA gene identified homologues of sdhC and sdhD; however, further sequencing by gene walking failed to identify additional sdh gene homologues. The sequence downstream of sdhD identified a partial gene, pep1, predicted to encode a protein > 35.3 kDa with 26.3% identity to a hypothetical Ehrlichia canis protein with no known function. The recently completed sequence of the A. phagocytophilum genome confirmed our findings and indicated that the sdhA and sdhB genes are duplicated in a tandem orientation, and located distant from the sdhC and sdhD genes. The expression of the A. phagocytophilum 16S rRNA, sdhC, and sdhD genes was examined by reverse transcriptase PCR which showed that these three genes are expressed as an operon. The pep1 gene was expressed independent of the 16S- sdhCD operon from a promoter between sdhD and pep1. Further analysis of the sdhA and sdhB genes suggested the tandem duplication of the genes in conserved and may be unique to the species A. phagocytophilum. While the conservation of the A. phagocytophilum Sdh proteins, including the residues required for heme- and quinone-binding by SdhC and SdhD, suggests these subunits form an active enzymatic complex, the unusual genomic arrangement and expression pattern of these genes support previous studies (rRNA, ftsZ) indicating that gene rearrangement and operon fragmentation are common in the genomes of Anaplasma and other obligate intracellular bacteria. OMB disclaimer: the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the CDC or the Department of Health and Human Services.

Age-dependent protein modifications and declining proteasome activity in the human lens

The proteasome is known to be the main enzymatic complex responsible for the intracellular degradation of altered proteins, and the age-related accumulation of modified lens proteins is associated to the formation of cataracts. The aim of this study was to determine whether the human lens proteasome becomes functionally impaired with age. The soluble and insoluble protein fractions of human lenses corresponding to various age-groups were characterized in terms of their levels of glyco-oxidative damage and found to show increasing anti-carboxymethyl-lysine immunoreactivity with age. Concomitantly, decreasing proteasome contents and peptidase activities were observed in the water-soluble fraction. The fact that peptidylglutamyl–peptide hydrolase activity is most severely affected with age suggests that specific changes are undergone by the proteasome itself. In particular, increasing levels of carboxymethylation were observed with age in the proteasome. It was concluded that the lower levels of soluble active enzymatic complex present in elderly lenses and the post-translational modifications affecting the proteasome may at least partly explain the decrease in proteasome activity and the concomitant accumulation of carboxymethylated and ubiquitinated proteins which occur with age.

Interaction of acid phosphatase with clays, organic molecules and organo-mineral complexes: kinetics and stability

The properties of synthetic active enzymatic complexes, simulating those usually present in soil environment, were investigated. Complexes were formed by the interaction of acid phosphatase with clays (montmorillonite and Al hydroxide), tannic acid and organo-mineral aggregates, obtained by mixing tannate, OH-Al species and/or montmorillonite. Immobilized acid phosphatase showed catalytic features quite different from those of the free enzyme. The presence of OH-Al species in the matrix generally resulted in an improvement of some enzymatic properties. A gain in activity of about 45 and 55% was observed for the complexes acid phosphatase–tannate–OH-Al species after thermal deactivation at 60°C and 2 h of exposure to proteinase K. High residual activities ranging from 17 to 61% and from 28 to 57% of the initial one were measured for complexes of the enzyme with inorganic and organic/organo-mineral matrices, respectively. In contrast, the association with a pure constituent such as montmorillonite and/or tannic acid gave rise to an immobilized enzyme, displaying a completely different catalytic behaviour. Compared to the free enzyme, acid phosphatase–montmorillonite and acid phosphatase–tannate complexes had a different pH-activity dependence and a higher and lower sensitivity to temperature and proteolysis, respectively.

MITOCHONDRIAL KINETICS DURING AMPHIBIAN ERYTHROPOIESIS RELATED TO HAEME SYNTHESIS

Flow cytometry, light and epifluorescence microscopies and transmission electron microscopy were used to follow the mitochondrial kinetics during amphibian erythropoiesis. A similar behaviour in response to the induction of anaemia was observed in the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. A high cellular activity was observed ten days after haemolytic anaemia induced by phenylhydrazine, based on the higher Rhodamine 123 uptake by the erythroid cells. In addition, the more intense expression of the mitochondrial enzyme cytochrome oxidase, isocitrate and succinic dehydrogenases were cytochemically detected at this stage. This suggests that erythroid cell mitochondria, at this time, could be in a more active functional state than at other stages. In both species, mitochondrial plasticity was observed during cell maturation. A progressive loss of oxidation–reduction enzyme expression seemed to follow changes at the mitochondrial cristae morphology, from transverse to longitudinal form, mainly at the 20th day of recovery from anaemia, possibly related to a natural loss of function. The presence of these mitochondrial enzymes in mitochondrion-like organelles also favours their participation in the haeme synthesis, although with a reduced expression, since this suggests the presence of a complete and active enzymatic complex in these modified organelles. This also supports the idea that all these organelles are mitochondria in distinct metabolic stages, and not mitochondrion-like organelles or haemosomes, as proposed by some authors.

Glycosylation of Asparagine-28 of Recombinant Staphylokinase with High-Mannose-type Oligosaccharides Results in a Protein with Highly Attenuated Plasminogen Activator Activity

The properties of recombinant staphylokinase (SakSTAR) expressed in Pichia pastoris cells have been determined. The single consensus N-linked oligosaccharide linkage site in SakSTAR (at Asn28 of the mature protein) was occupied in approximately 50% of the expressed protein with high-mannose-type oligosaccharides. The majority of these glycans ranged in polymerization state from Man8GlcNAc2 to Man14GlcNAc2, with the predominant species being Man10GlcNAc2 and Man11GlcNAc2. Glycosylated SakSTAR (SakSTARg) did not differ from its aglycosyl form in its aggregation state in solution, its thermal denaturation properties, its ability to form a complex with human plasmin (hPm), the amidolytic properties of the respective SakSTAR-hPm complexes, or its ability to liberate the amino-terminal decapeptide required for formation of a functional SakSTAR-hPm plasminogen activator complex. However, this latter complex with SakSTARg showed a greatly reduced ability to activate human plasminogen (hPg) as compared with the same complex with the aglycosyl form of SakSTAR. We conclude that glycosylation at Asn28 does not affect the structural properties of SakSTAR or its ability to participate in the formation of an active enzymatic complex with hPm, but it is detrimental to the ability of the SakSTAR-hPm complex to serve as a hPg activator. This is likely due to restricted access of hPg to the active site of the SakSTARg-hPm complex.

INTERACTIONS OF ACID PHOSPHATASE WITH CLAYS, ORGANIC MOLECULES AND ORGANO-MINERAL COMPLEXES1

Synthetic active enzymatic complexes have been formed by the interaction of acid phosphatase with clays (montmorillonite and chlorites), organic molecules, and organo-mineral associations. The inhibition of each component on the activity of the free enzyme and the residual activity of the enzymatic complexes have both been determined. Clays and tannic acid displayed various inhibitory effects on acid phosphatase activity. Montmorillonite and aluminum hydrous oxide showed the highest (70%) and the lowest (<5%) inhibitory influence, respectively. The covering of montmorillonite surfaces with OH-Al species increased not only the amount but also the residual activity of adsorbed acid phosphatase molecules. When 18 meq of Al g -1 clay were adsorbed on montmorillonite surfaces, the residual activity of adsorbed enzyme increased up to 54% of that added initially. The activity retained by tannate-acid phosphatase complexes depended on incubation time and tannic acid/enzyme ratios. This suggests that a complexation between tannic acid and enzyme molecules, rather than a polymerization process of tannic acid involving acid phosphatase, took place. The presence of OH-Al species during the interaction between the two components favored the entrapment of more enzymatic molecules at higher activity levels.

Kinetic Behavior of Synthetic Organo‐ and Organo‐Mineral‐Urease Complexes

AbstractA study was conducted for characterizing the properties and the catalytic behavior of enzymatic complexes that are considered analogues of those usually present in soil. Active enzymatic complexes were formed at 10°C by interaction for 1 h of urease and tannic acid with and without Fe ions and OH‐Al polymers. The enzymatic complexes obtained in the presence of Fe3+ ions and OH‐Al species showed higher activity levels than urease‐tannate complexes. The pH optimum of immobilized urease did not differ significantly from that of free urease, except for the tannate complexes, which showed a pH optimum slightly shifted upwards by ≈0.5 units. Immobilized urease displayed the highest activity in a wider range of temperatures (60–80°C) compared with the free enzyme. The free enzyme showed a marked reduction in activity above 70°C. Higher values of energy of activation (Ea), enthalpy of activation (ΔHa), and the kinetic parameter (Km) were obtained for urease immobilized on tannate molecules. On the contrary, the complexes obtained with Fe3+ and OH‐Al species displayed kinetic and thermodynamic parameters similar to those of the free enzyme. Immobilized urease showed a catalytic behavior similar to the free enzyme, when the interaction with tannic acid occurred in the presence of Fe3+ ions and OH‐Al polymers. Evidently, both Fe ions and OH‐Al species favored the formation of enzymatic complexes in which conformational and catalytic features of the enzyme remained practically unchanged.

Formation and Activity of Urease‐Tannate Complexes Affected by Aluminum, Iron, and Manganese

AbstractThe formation of active enzymatic complexes, obtained by the interaction of urease, tannic acid, and soluble and insoluble species of Al, Fe, or Mn, was studied. Tannic acid strongly inhibited urease activity (inhibition constant, Ki = 0.040 mM). The dependence of kinetic parameters (Vmax and Km) on tannic acid concentration suggests a pure uncompetitive inhibition mechanism. Soluble and insoluble urease‐tannate complexes were formed by increasing both the contact time and the tannic acid/urease ratios. Soluble (FeCl3, MnCl2, and OH‐Al polymers) and insoluble (Fe2O3 and MnO2) inorganic components differently influenced the interaction of urease with tannic acid molecules and the residual activity of the immobilized enzyme. An increased removal of urease molecules from solution as well as an enhanced residual enzymatic activity of urease complexes was observed in the presence of all the additives. Soluble species (FeCl3 and OH‐Al polymers) probably acted as flocculating agents or promoted the formation of more active enzymatic complexes. On the contrary, MnO2 showed a predominant polymerizing action on tannate molecules that gave rise to insoluble aggregates occluding urease molecules. Adsorption, as opposed to polymerization, probably occurred in the presence of Fe2O3.

Interactions of invertase with tannic acid, hydroxy-aluminium (OH-A1) species or montmorillonite

Interactions between invertase, tannic acid and hydroxy-aluminium (OH-A1) polymers were investigated with regard to their influence on enzyme activity and the formation of active enzymatic complexes. OH-A1 species slightly inhibited invertase activity (inhibition constant, K i = 133 him) whereas a marked inhibition (>90%) was detected at a tannic acid concentration greater than 1.25 mM. The V max and K m values indicated a pure non-competitive and a mixed-type inhibition mechanism of OH-A1 species and tannic acid, respectively. Depending on the incubation time and the tannic acid-to-enzyme ratio, the interaction between invertase and tannic acid yielded both soluble and insoluble complexes, which displayed reduced activity levels. The decrease of invertase activity was a function of both the tannic acid-to-invertase ratios and the contact time. Bonding between invertase and tannic acid as well as the residual activity of the immobilized enzyme were greatly increased by the presence of OH-A1 polymers during the complexation process. Many more active invertase molecules were removed from solution and an enhanced precipitation of active invertase-tannic acid complexes was observed. Clearly not only OH-A1 ions facilitated flocculation of tannate-invertase complexes with different structural characteristics but their interaction with tannic acid molecules gave rise to Al precipitation products having a different charge and sorption sites. Furthermore, an invertase-OH-Al-tannic acid complex was anchored on montmorillonite surfaces and the complex formed showed a relatively high enzymatic activity. OH-A1 species acting as bridges between tannate molecules and montmorillonite surfaces also facilitated the immobilization of soluble invertase-OH-Al-tannate complexes.

The involvement of oxygen radicals in microbicidal mechanisms of leukocytes and macrophages

Phagocytic leukocytes generate large amounts of reactive oxygen compounds during and after phagocytosis of micro-organisms. These compounds are essential for the killing of a wide variety of microbes. The enzyme responsible for this process is NADPH:O2 oxidoreductase (NADPH oxidase), which utilizes the reduction equivalents of NADPH to reduce atmospheric oxygen to superoxide (O2-.). Subsequently, superoxide is converted by the leukocytes to other reactive compounds, such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and N-chloramines (RNCl). Each of these compounds has potent microbicidal properties. Under resting, non-phagocytizing conditions, phagocytes do not produce reactive oxygen compounds. However, within 15-30 sec after binding of micro-organisms to cell surface receptors, superoxide generation starts. This phenomenon is called the respiratory burst. This phenomenon is called the respiratory burst. The activation of the NADPH oxidase is caused by the assembly of components of this enzyme into an active complex. Under resting conditions, at least three components reside in the cytoplasm and at least two are located in the plasma membrane. Activation of the NADPH oxidase results in translocation of cytosolic components to the plasma membrane and formation of an active enzymatic complex in the plasma membrane.