Identification of oxidative modifications of hemopexin and their predicted physiological relevance.

Hemopexin protects against heme toxicity in hemolytic diseases and conditions, sepsis, and sickle cell disease. This protection is sustained by heme-hemopexin complexes in biological fluids that resist oxidative damage during heme-driven inflammation. However, apo-hemopexin is vulnerable to inactivation by reactive nitrogen (RNS) and oxygen species (ROS) that covalently modify amino acids. The resultant nitration of amino acids is considered a specific effect reflecting biological events. Using LC-MS, we discovered low endogenous levels of tyrosine nitration in the peptide YYCFQGNQFLR in the heme-binding site of human hemopexin, which was similarly nitrated in rabbit and rat hemopexins. Immunoblotting and selective reaction monitoring were used to quantify tyrosine nitration of in vivo samples and when hemopexin was incubated in vitro with nitrating nitrite/myeloperoxidase/glucose oxidase. Significantly, heme binding by hemopexin declined as tyrosine nitration proceeded in vitro Three nitrated tyrosines reside in the heme-binding site of hemopexin, and we found that one, Tyr-199, interacts directly with the heme ring D propionate. Investigating the oxidative modifications of amino acids after incubation with tert-butyl hydroperoxide and hypochlorous acid in vitro, we identified additional covalent oxidative modifications on four tyrosine residues and one tryptophan residue of hemopexin. Importantly, three of the four modified tyrosines, some of which have more than one modification, cluster in the heme-binding site, supporting a hierarchy of vulnerable amino acids. We propose that during inflammation, apo-hemopexin is nitrated and oxidated in niches of the body containing activated RNS- and ROS-generating immune and endothelial cells, potentially impairing hemopexin's protective extracellular antioxidant function.

Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis.

Drosophila DBT and vertebrate CKIε/δ phosphorylate the period protein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBT(C/ala)) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIδ, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBT(C/ala) did not affect circadian behavior differently from wild-type DBT (DBT(WT)), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT(WT) protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT(C/ala) did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.

Drosophila spaghetti and doubletime link the circadian clock and light to caspases, apoptosis and tauopathy.

While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in the optic lobes.

Noncanonical FK506-binding protein BDBT binds DBT to enhance its circadian function and forms foci at night.

The kinase DOUBLETIME is a master regulator of the Drosophila circadian clock, yet the mechanisms regulating its activity remain unclear. A proteomic analysis of DOUBLETIME interactors led to the identification of an unstudied protein designated CG17282. RNAi-mediated knockdown of CG17282 produced behavioral arrhythmicity and long periods and high levels of hypophosphorylated nuclear PERIOD and phosphorylated DOUBLETIME. Overexpression of DOUBLETIME in flies suppresses these phenotypes and overexpression of CG17282 in S2 cells enhances DOUBLETIME-dependent PERIOD degradation, indicating that CG17282 stimulates DOUBLETIME's circadian function. In photoreceptors, CG17282 accumulates rhythmically in PERIOD- and DOUBLETIME-dependent cytosolic foci. Finally, structural analyses demonstrated CG17282 is a noncanonical FK506-binding protein with an inactive peptide prolyl-isomerase domain that binds DOUBLETIME and tetratricopeptide repeats that may promote assembly of larger protein complexes. We have named CG17282 BRIDE OF DOUBLETIME and established it as a mediator of DOUBLETIME's effects on PERIOD, most likely in cytosolic foci that regulate PERIOD nuclear accumulation.

Drosophila and vertebrate casein kinase Idelta exhibits evolutionary conservation of circadian function.

Mutations lowering the kinase activity of Drosophila Doubletime (DBT) and vertebrate casein kinase Iepsilon/delta (CKIepsilon/delta) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckI short-period mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period (PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKIepsilon/delta. Moreover, expression of either Drosophila DBT or the vertebrate CKIdelta kinase carrying the Drosophila dbt(S) or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKIdelta carrying the dbt(L) mutation does not lengthen circadian rhythms, while Drosophila DBT(L) does. Different effects of the dbt(S) and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKIdelta and of the functions affected by their period-shortening mutations.

Drosophila DBT lacking protein kinase activity produces long-period and arrhythmic circadian behavioral and molecular rhythms.

A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBT(K/R)) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBT(K/R) antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered "set point" for period length that is not altered by overexpression. Overexpression of the DBT(K/R) produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods.

Drosophila doubletime mutations which either shorten or lengthen the period of circadian rhythms decrease the protein kinase activity of casein kinase I.

In both mammals and fruit flies, casein kinase I has been shown to regulate the circadian phosphorylation of the period protein (PER). This phosphorylation regulates the timing of PER's nuclear accumulation and decline, and it is necessary for the generation of circadian rhythms. In Drosophila melanogaster, mutations affecting a casein kinase I (CKI) ortholog called doubletime (dbt) can produce short or long periods. The effects of both a short-period (dbt(S)) and long-period (dbt(L)) mutation on DBT expression and biochemistry were analyzed. Immunoblot analysis of DBT in fly heads showed that both the dbt(S) and dbt(L) mutants express DBT at constant levels throughout the day. Glutathione S-transferase pull-down assays and coimmunoprecipitation of DBT and PER showed that wild-type DBT, DBT(S), and DBT(L) proteins can bind to PER equivalently and that these interactions are mediated by the evolutionarily conserved N-terminal part of DBT. However, both the dbt(S) and dbt(L) mutations reduced the CKI-7-sensitive kinase activity of an orthologous Xenopus laevis CKIdelta expressed in Escherichia coli. Moreover, expression of DBT in Drosophila S2 cells produced a CKI-7-sensitive kinase activity which was reduced by both the dbt(S) and dbt(L) mutations. Thus, lowered enzyme activity is associated with both short-period and long-period phenotypes.