Protein substrate discrimination in the quiescin sulfhydryl oxidase (QSOX) family

This work explores the substrate specificity of the quiescin sulfhydryl oxidase (QSOX) family of disulfide-generating flavoenzymes to provide enzymological context for investigation of the physiological roles of these facile catalysts of oxidative protein folding. QSOX enzymes are generally unable to form disulfide bonds within well-structured proteins. Use of a temperature-sensitive mutant of ubiquitin-conjugating enzyme 4 (Ubc4') as a model substrate shows that QSOX activity correlates with the unfolding of Ubc4' monitored by circular dichroism. Fusion of Ubc4' with the more stable glutathione-S-transferase domain demonstrates that QSOX can selectively introduce disulfides into the less stable domain of the fusion protein. In terms of intermolecular disulfide bond generation, QSOX is unable to cross-link well-folded globular proteins via their surface thiols. However, the construction of a septuple mutant of RNase A, retaining a single cysteine residue, demonstrates that flexible protein monomers can be directly coupled by the oxidase. Steady- and pre-steady-state kinetic experiments, combined with static fluorescence approaches, indicate that while QSOX is an efficient catalyst for disulfide bond formation between mobile elements of structure, it does not appear to have a significant binding site for unfolded proteins. These aspects of protein substrate discrimination by QSOX family members are rationalized in terms of the stringent steric requirements for disulfide exchange reactions.     

Novel tricyclic indeno[2,1-d]pyrimidines with dual antiangiogenic and cytotoxic activities as potent antitumor agents

We designed, synthesized and evaluated 13 novel tricyclic indeno[2,1-d]pyrimidines as RTK inhibitors. These analogues were synthesized via a Dieckmann condensation of 1,2-phenylenediacetonitrile followed by cyclocondensation with guanidine carbonate to afford the 2-amino-3,9-dihydro-indeno[2,1-d]pyrimidin-4-one. Sulfonation of the 4-position followed by displacement with appropriately substituted anilines afforded the target compounds. These compounds were potent inhibitors of platelet-derived growth factor receptor β (PDGFRβ) and inhibited angiogenesis in the chicken embryo chorioallantonic membrane (CAM) assay compared to standards. In addition, compound 7 had a two digit nanomolar GI(50) against nine tumor cell lines, a submicromolar GI(50) against 29 of other tumor cell lines in the preclinical NCI 60 tumor cell line panel. Compound 7 also demonstrated significant in vivo inhibition of tumor growth and angiogenesis in a B16-F10 syngeneic mouse melanoma model.     

Oxidant-induced cell-cycle delay in Saccharomyces cerevisiae: the involvement of the SWI6 transcription factor

Cells treated with low doses of linoleic acid hydroperoxide (LoaOOH) exhibit a cell-cycle delay that may provide a mechanism to overcome oxidative stress. Strains sensitive to LoaOOH from the genome-wide deletion collection were screened to identify deletants in which the cell-cycle delay phenotype was reduced. Forty-seven deletants were identified that were unable to mount the normal delay response, implicating the product of the deleted gene in the oxidant-mediated cell-cycle delay of the wild-type. Of these genes, SWI6 was of particular interest due to its role in cell-cycle progression through Start. The swi6 deletant strain was delayed on entry into the cell cycle in the absence of an oxidant, and oxidant addition caused no further delay. Transforming the swi6 deletant with SWI6 on a plasmid restored the G1 arrest in response to LoaOOH, indicating that Swi6p is involved in oxidant sensing leading to cell division delay. Micro-array studies identified genes whose expression in response to LoaOOH depended on SWI6. The screening identified 77 genes that were upregulated in the wild-type strain and concurrently downregulated in the swi6 deletant treated with LoaOOH. These data show that functions such as heat shock response, and glucose transport are involved in the response.     

Acyl-CoA dehydrogenases. A mechanistic overview.

Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the alpha,beta-dehydrogenation of fatty acid acyl-CoA conjugates. While they differ widely in their specificity, they share the same basic chemical mechanism of alpha,beta-dehydrogenation. Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms. Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein. The mechanism of alpha,beta-dehydrogenation is viewed as a process in which the substrate alphaC-H and betaC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO- as an H+, the second being transferred as a hydride to the flavin N(5) position. Hereby the pKa of the substrate alphaC-H is lowered from > 20 to approximately 8 by the effect of specific hydrogen bonds. Concomitantly, the pKa of Glu376-COO- is also raised to 8-9 due to the decrease in polarity brought about by substrate binding. The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates. A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein. These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases.     

Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells

The objective of this study was to investigate the influence of dynamic compressive loading on chondrogenesis of mesenchymal stem cells (MSCs) in the presence of TGF-β3. Isolated porcine MSCs were suspended in 2% agarose and subjected to intermittent dynamic compression (10% strain) for a period of 42 days in a dynamic compression bioreactor. After 42 days in culture, the free-swelling specimens exhibited more intense alcian blue staining for proteoglycans, while immunohistochemical analysis revealed increased collagen type II immunoreactivity. Glycosaminoglycan (GAG) content increased with time for both free-swelling and dynamically loaded constructs, and by day 42 it was significantly higher in both the core (2.5 ± 0.21%w/w vs. 0.94 ± 0.03%w/w) and annulus (1.09 ± 0.09%w/w vs. 0.59 ± 0.08%w/w) of free-swelling constructs compared to dynamically loaded constructs. This result suggests that further optimization is required in controlling the biomechanical and/or the biochemical environment if such stimuli are to have beneficial effects in generating functional cartilaginous tissue.     

Human quiescin-sulfhydryl oxidase, QSOX1: probing internal redox steps by mutagenesis

The flavoprotein quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active toward reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower k cat/ K m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FAD-proximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain.     

Purine and pyrimidine metabolism during the partial drying treatment of white spruce (Picea glauca) somatic embryos

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of ¹⁴C-labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO₂ and β - ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination.     

Formation of pentosidine during nonenzymatic browning of proteins by glucose. Identification of glucose and other carbohydrates as possible precursors of pentosidine in vivo

A fluorescent compound has been detected in proteins browned during Maillard reactions with glucose in vitro and shown to be identical to pentosidine, a pentose-derived fluorescent cross-link formed between arginine and lysine residues in collagen (Sell, D. R., and Monnier, V. M. (1989) J. Biol. Chem. 264, 21597-21602). Pentosidine was the major fluorophore formed during nonenzymatic browning of ribonuclease and lysozyme by glucose, but accounted for less than 1% of non-disulfide cross-links in protein dimers formed during the reaction. Pentosidine was formed in greatest yields in reactions of pentoses with lysine and arginine in model systems but was also formed from glucose, fructose, ascorbate, Amadori compounds, 3-deoxyglucosone, and other sugars. Pentosidine was not formed from peroxidized polyunsaturated fatty acids or malondialdehyde. Its formation from carbohydrates was inhibited under nitrogen or anaerobic conditions and by aminoguanidine, an inhibitor of advanced glycation and browning reactions. Pentosidine was detected in human lens proteins, where its concentration increased gradually with age, but it did not exceed trace concentrations (less than or equal to 5 mumol/mol lysine), even in the 80-year-old lens. Although its precise carbohydrate source in vivo is uncertain and it is present in only trace concentrations in tissue proteins, pentosidine appears to be a useful biomarker for assessing cumulative damage to proteins by nonenzymatic browning reactions with carbohydrates.     

Soluble and cell-wall peroxidases and auxin destruction in normal and habituated tobacco callus

Summary A comparison of the soluble and cell-wall-bound isoperoxidases of normal auxin-requiring and auxin-independent (habituated) tobacco callus revealed that normal tissues contained a higher level of isoperoxidases. There were also qualitative differences in these isoperoxidases. Partially purified soluble and ionically bound isoperoxidases of normal callus likewise exhibited higher auxin-oxidase activities. Normal tissues also were found to contain higher levels of auxin-oxidase inhibitors (auxin protectors). Overall, however, the data indicate that there is a higher rate of auxin destruction in normal tobacco callus than in habituated tissue. This presumably leads to insufficient endogenous auxin for growth. This study was supported in part by grants to T.G. from the Center IRSIA d'Etude de la Reproduction végétale and the FRFC Contract No. 2.9009. it Was carried out while T.A.T. was the holder of a senior Fellowship under the NATO senior Scientists Program.     

Periodicity in Atlantic salmon Salmo salar L. smolt migration

Behavioural and physiological mechanisms postulated for the control of downstream migration of Atlantic salmon smolts are reviewed briefly, and some new evidence is presented for their refusal to undergo sustained swimming. Although these mechanisms imply passive displacement as the primary means of emigration, it is likely that active components must also exist as the rates of travel of smolts through loch systems are only slightly slower than those recorded for river systems. The timing of these movements within 24 h periods is reviewed and it is shown that the predominantly nocturnal emigration pattern is evident on occasions in alevin, fry and parr stages also. Thus at migration the diel periodicity probably represents a seasonal locomotor rhythm which, under changed behavioural and physiological circumstances, results in downstream displacement.