Annexin A1 is regulated by domains cross-talk through post-translational phosphorylation and SUMOYlation

Mouse prostate membrane-associated proteins of the annexin family showed changes in SUMOylation during androgen treatment. Among these the calcium-binding annexin A1 protein (ANXA1) was chosen for further characterization given its role in protein secretion and cancer. SUMOylation of ANXA1 was confirmed by overexpressing SUMO-1 in LNCaP cells. Site-directed mutagenesis indicated that K257 located in a SUMOylation consensus motif in the C-terminal calcium-binding DA3 repeat domain is SUMOylated. Mutation of the N-terminal Y21 decreased markedly the SUMOylation signal while EGF stimulation increased ANXA1 SUMOylation. A structural analysis of ANXA1 revealed that K257 is located in a hot spot where Ca^2 + and SUMO-1 bind and where a nuclear export signal and a polyubiquitination site are also present. Also, Y21 is buried inside an α-helix structure in the Ca^2 +-free conformation implying that Ca^2 + binding, and the subsequent expelling of the N-terminal α-helix in a disordered conformation, is permissive for its phosphorylation. These results show for the first time that SUMOylation can be regulated by an external signal (EGF) and indicate the presence of a cross-talk between the N-terminal and C-terminal domains of ANXA1 through post-translational modifications.     

Identification of human fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) as a novel mitochondrial acylpyruvase

The human fumarylacetoacetate hydrolase (FAH) domain-containing protein 1 (FAHD1) is part of the FAH protein superfamily, but its enzymatic function is unknown. In the quest for a putative enzymatic function of FAHD1, we found that FAHD1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. Conserved amino acids Asp-102 and Arg-106 of FAHD1 were found important for its catalytic activity, and Mg(2+) was required for maximal enzyme activity. FAHD1 was found expressed in all tested murine tissues, with highest expression in liver and kidney. FAHD1 was also found in several human cell lines, where it localized to mitochondria. In summary, the current work identified mammalian FAHD1 as a novel mitochondrial enzyme with acylpyruvate hydrolase activity.     

Analysis and Phylogeny of Small Heat Shock Proteins from Marine Viruses and Their Cyanobacteria Host

Small heat shock proteins (sHSPs) are oligomeric stress proteins characterized by an α-crystallin domain (ACD) surrounded by a N-terminal arm and C-terminal extension. Publications on sHSPs have reported that they exist in prokaryotes and eukaryotes but, to our knowledge, not in viruses. Here we show that sHSPs are present in some cyanophages that infect the marine unicellular cyanobacteria, Synechococcus and Prochlorococcus. These phage sHSPs contain a conserved ACD flanked by a relatively conserved N-terminal arm and a short C-terminal extension with or without the conserved C-terminal anchoring module (CAM) L-X-I/V, suggested to be implicated in the oligomerization. In addition, cyanophage sHSPs have the signature pattern, P-P-[YF]-N-[ILV]-[IV]-x(9)-[EQ], in the predicted β2 and β3 strands of the ACD. Phylogenetically, cyanophage sHSPs form a monophyletic clade closer to bacterial class A sHSPs than to cyanobacterial sHSPs. Furthermore, three sHSPs from their cellular host, Synechococcus, are phylogenetically close to plants sHSPs. Implications of evolutionary relationships between the sHSPs of cyanophages, bacterial class A, cyanobacteria, and plants are discussed.     

Zebrafish (Danio rerio) fed vitamin E-deficient diets produce embryos with increased morphologic abnormalities and mortality

Vitamin E (α-tocopherol) is required to prevent fetal resorption in rodents. To study α-tocopherol's role in fetal development, a nonplacental model is required. Therefore, the zebrafish, an established developmental model organism, was studied by feeding the fish a defined diet with or without added α-tocopherol. Zebrafish (age, 4-6 weeks) were fed the deficient (E-), sufficient (E+) or lab diet up to 1 years. All groups showed similar growth rates. The exponential rate of α-tocopherol depletion up to ~80 day in E- zebrafish was 0.029±0.006 nmol/g, equivalent to a depletion half-life of 25±5 days. From age ~80 days, the E- fish (5±3 nmol/g) contained ~50 times less α-tocopherol than the E+ or lab diet fish (369±131 or 362±107, respectively; P<.05). E-depleted adults demonstrated decreased startle response suggesting neurologic deficits. Expression of selected oxidative stress and apoptosis genes from livers isolated from the zebrafish fed the three diets were evaluated by quantitative polymerase chain reaction and were not found to vary with vitamin E status. When E-depleted adults were spawned, they produced viable embryos with depleted α-tocopherol concentrations. The E- embryos exhibited a higher mortality (P<.05) at 24 h post-fertillization and a higher combination of malformations and mortality (P<.05) at 120 h post-fertillization than embryos from parents fed E+ or lab diets. This study documents for the first time that vitamin E is essential for normal zebrafish embryonic development.     

Tissue-specific expression of heat shock proteins of the mouse in the absence of stress

The steady-state levels of four members of the heat shock proteins families (HSP84, HSC73, HSP71, and HSP25) were examined by immunoblot analysis of several different tissues of young and adult mice in the absence of stress. These hsps were detected in all tissues but their level was variable. The levels of HSC73 and HSP84 varied only slightly between different tissues in either young or adult mice, with the exception of skin where these hsps were found in reduced amounts. In contrast, the stress-inducible member of the HSP70 family, HSP71, was found to be expressed in all tissues but in amounts which differed by as much as two orders of magnitude between tissues. In general, the levels of both HSP71 and HSP25 were found to be tissue dependent, with higher levels found in tissues such as stomach, intestine, colon and bladder, tissues which are exposed to toxic environmental or metabolic products, and which may concentrate these substances by water resorption and/or be exposed to them for longer periods. The levels of HSP71 and HSP25 were generally positively correlated both in young and adult mice although this correlation was not found in certain tissues such as kidney, testes, and bone. Tissues of young mice contained lower amounts of HSP25 and HSP71 than were found in the same tissues from adults. We conclude that hsps are expressed in all tissues of the mouse in the absence of stress and that some organs, particularly those exposed to potentially toxic metabolites, show a higher level of expression of HSP71 and HSP25. © 1993Wiley-Liss, Inc.     

Oligomerization and chaperone-like activity of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> small heat shock protein DmHsp27 and three arginine mutants in the alpha-crystallin domain

The small Hsp DmHsp27 from Drosophila melanogaster is one of the few small heat shock proteins (sHsps) found within the nucleus. We report that its dimerization is independent of disulfide bond formation and seems to rely on salt bridges. Unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations at highly conserved arginine residues in mammalian sHsps have been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131, and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild-type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G, and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild-type DmHsp27 was lower than that of R122G and R131G mutants. Altogether, the results characterize wild-type DmHsp27 and its alpha-crystallin domain (ACD) arginine mutants and may give insight into protection mechanism of sHsps.     

A missense mutation (Q279R) in the Fumarylacetoacetate Hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation

Background  

Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was found in tumor regions while a healthy region contained enzyme-expressing nodules.