The three-dimensional structure of the ZAP-70 kinase domain in complex with staurosporine: implications for the design of selective inhibitors

The ZAP-70 tyrosine kinase plays a critical role in T cell activation and the immune response and therefore is a logical target for immunomodulatory therapies. Although the crystal structure of the tandem Src homology-2 domains of human ZAP-70 in complex with a peptide derived from the zeta subunit of the T cell receptor has been reported (Hatada, M. H., Lu, X., Laird, E. R., Green, J., Morgenstern, J. P., Lou, M., Marr, C. S., Phillips, T. B., Ram, M. K., Theriault, K., Zoller, M. J., and Karas, J. L. (1995) Nature 377, 32-38), the structure of the kinase domain has been elusive to date. We crystallized and determined the three-dimensional structure of the catalytic subunit of ZAP-70 as a complex with staurosporine to 2.3 A resolution, utilizing an active kinase domain containing residues 327-606 identified by systematic N- and C-terminal truncations. The crystal structure shows that this ZAP-70 kinase domain is in an active-like conformation despite the lack of tyrosine phosphorylation in the activation loop. The unique features of the ATP-binding site, identified by structural and sequence comparison with other kinases, will be useful in the design of ZAP-70-selective inhibitors.     

Melatonin,immune function and aging

Aging is associated with a decline in immune function (immunosenescence), a situation known to correlate with increased incidence of cancer, infectious and degenerative diseases. Innate, cellular and humoral immunity all exhibit increased deterioration with age. A decrease in functional competence of individual natural killer (NK) cells is found with advancing age. Macrophages and granulocytes show functional decline in aging as evidenced by their diminished phagocytic activity and impairment of superoxide generation. There is also marked shift in cytokine profile as age advances, e.g., CD3+ and CD4+ cells decline in number whereas CD8+ cells increase in elderly individuals. A decline in organ specific antibodies occurs causing reduced humoral responsiveness. Circulating melatonin decreases with age and in recent years much interest has been focused on its immunomodulatory effect. Melatonin stimulates the production of progenitor cells for granulocytes-macrophages. It also stimulates the production of NK cells and CD4+ cells and inhibits CD8+ cells. The production and release of various cytokines from NK cells and T-helper lymphocytes also are enhanced by melatonin. Melatonin presumably regulates immune function by acting on the immune-opioid network, by affecting G protein-cAMP signal pathway and by regulating intracellular glutathione levels. Melatonin has the potential therapeutic value to enhance immune function in aged individuals and in patients in an immunocompromised state.     

Hormonal Effects on Calcium Metabolism in Crustacea

Cyclic shifts of calcium in the exoskeleton and soft tissues,as they are related to the intermolt cycle in crayfish, arereviewed. Regulatory factors, derived from the eyestalk, influencelevels of exoskeletal calcium; eyestalk extracts prepared fromanimals in premolt decrease shell calcium, while reciprocallyextracts from animals in intermolt increase it when these hormonalsources are injected into animals in the premolt stage (D₀-D₄). In addition, premolt eyestalk extract results in an increasein gastrolith calcium. In the exchange of calcium between theanimal and its environment there is evidence for differentialdepositionof recently available calcium in the exoskeleton. Further, intermoltand early premolt animals maintained in Ca⁴⁵-labelled waterfor 15 days concentrate it 4 and 3—fold in the exoskeletonand stomach, respectively. However, removal of a molt-inhibitingfactor through ablation of eyestalks results in a 20 and 40—foldincrease in incorporation inthese same sites relative to environmentalconcentrations. Treatment with mammalian parathyroid extract mobilizes bothexoskeletal and gastric calciumand leads to a rise in bloodcalcium. However, there is little or no effect on levels ofexoskeletal citric acid. Further, citric acid is higher in thecrayfish carapace during stage C, the period of mineralization,than in stage D, the period of demineralization. There are both similarities and differences between the effectsof crustacean and mammalianregulating factors with respect tothe direction and extent of mineralization. Biochemical studiesshould elucidate the mechanisms regulated by these hormones.     

The partition matrix: exploring variable phylogenetic signals along nucleotide sequence alignments

The partition matrix is a graphical tool for comparative analysis of nucleotide sequences following alignment. It is particularly useful for investigating the divergent phylogenies of sequence regions undergoing reticulate evolution. A partition matrix is generated by determining the consistency of the parsimoniously informative sites in a set of aligned sequences with the binary partitions inferred from the sequences. Since the linear order of sites is maintained, the matrix can be used to assess whether the distribution of sites either supporting or conflicting with particular partitions changes along the length of the alignment. The usefulness of the matrix in allowing visual identification of differences in evolutionary history among regions depends on the order in which partitions are shown; several suitable ordering schemes are proposed. We demonstrate the use of the partition matrix in interpreting the evolution of the pseudoautosomal boundary region on the sex chromosome of catarrhine primates. Its routine use should help to avoid attempts to derive single phylogenies from sequences whose evolution has been reticulate and to identify the gene conversion or recombination events underlying the reticulation. The method is relatively fast. It is exploratory, and it can form the basis for more formal analysis, which we discuss.      

Positive selection and sequence rearrangements generate extensive polymorphism in the gamete recognition protein bindin

Bindin is a gamete recognition protein of sea urchins that mediates species-specific attachment of sperm to an egg-surface receptor during fertilization. Sequences of bindin from closely related urchins show fixed species-specific differences. Within species, highly polymorphic bindin alleles result from point substitution, insertion/deletion, and recombination. Since speciation, positive selection favoring allelic variants has generated diversity in bindin polypeptides. Intraspecific bindin variation can be tolerated by the egg receptor, which suggests functional parallels between this system and other flexible recognition systems, including immune recognition. These results show that polymorphism in mate recognition loci required for rapid evolution of sexual isolation can arise within natural populations.      

Mass density contrast in relation to the feeding currents in calanoid copepods

Theoretical analyses show that positively buoyant copepods areable to generate feeding currents by adopting upside-down bodypositions and pushing water upward. Thus, the excess buoyancyacting on the copepods will be balanced and cone-shaped feedingcurrents generated to transport water to the capture areas.The intensities of the feeding currents, which can be measuredin the present modeling study by calculating the volumetricflux going through the capture areas, are proportional to themass density contrasts between the copepods and the ambientseawater. The mass density contrasts may vary spatially andtemporally depending on copepod body contents and on the propertiesof the seawater immediately surrounding them. We focus on thecase where the mass density contrast between a wax ester-richcopepod and its ambient seawater can vary strongly with depthbecause wax esters are more compressible and 6–10 timesmore thermally expansible than seawater. These theoretical analysesshow that the intensities of the feeding currents generatedby wax ester-rich copepods vary strongly with depth. Our conclusionsfrom these theoretical analyses need to be tested by directobservations. This paper is one of six on the subject of the role of zooplanktonpredator–prey interactions in structuring plankton communities.     

Over-expression of human type I (placental) 3β-hydroxy-5-ene-steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus

Human type I placental 3β-hydroxy-5-ene-steroid dehydrogenase/steroid 5→4-ene-isomerase (3β-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3β-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3β-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3β-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3β-HSD substrate, 5-androstan-3β-o1-17-one, in the Sf-9 cell homogenate (K_m = 17.9 μM) and placental microsomes (K_m = 16.7 μM). The 3β-HSD activity (V_max = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (V_max = 9.1 nmol/min/mg). The K_m values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (K_m = 14.7 μM) and placental microsomes (K_m = 14.4 μM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (V_max = 25.7 nmol/min/mg) relative to placenta (V_max = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3β-HSD/isomerase in human placenta.     

Affinity alkylation of human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase by 2 alpha-bromoacetoxyprogesterone: evidence for separate dehydrogenase and isomerase sites on one protein

We have copurified human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH2-terminal sequencing data. The affinity alkylator, 2 alpha-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C21 and C19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations (50/1-20/1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity (t1/2 = 1.5-3.7 min) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepiandrosterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2 alpha-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism (k3' = 160.9 l/mol.s), while the alkylator inactivates isomerase by a unimolecular mechanism (Ki = 0.14 mM, k3 = 0.013 s-1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2 alpha-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD+ protects neither. From Dixon analysis, NADH competitively inhibits NAD+ reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD+ and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C21 and C19 isomerase activities at a second site on the same protein.