Ubiquitin ligase Ufd2 is required for efficient degradation of Mps1 kinase

Ufd2 is a U-box-containing ubiquitylation enzyme that promotes ubiquitin chain assembly on substrates. The physiological function of Ufd2 remains poorly understood. Here, we show that ubiquitylation and degradation of the cell cycle kinase Mps1, a known target of the anaphase-promoting complex E3, require Ufd2 enzyme. Yeast cells lacking UFD2 exhibit altered chromosome stability and several spindle-related phenotypes, expanding the biological function of Ufd2. We demonstrate that Ufd2-mediated Mps1 degradation is conserved in humans. Our results underscore the significance of Ufd2 in proteolysis and further suggest that Ufd2-like enzymes regulate far more substrates than previously envisioned.     

Reovirus infection or ectopic expression of outer capsid protein micro1 induces apoptosis independently of the cellular proapoptotic proteins Bax and Bak

Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein μ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed μ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of μ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for μ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of μ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing μ1, indicating that caspases were not required. Additionally, μ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax(-/-)Bak(-/-) MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak.     

Human breast cancer histoid: an in vitro 3-dimensional co-culture model that mimics breast cancer tissue

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 μm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.     

Reactive oxygen species regulate nucleostemin oligomerization and protein degradation

Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H(2)O(2)) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H(2)O(2) impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-L-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.     

Acireductone dioxygenase 1 (ARD1) is an effector of the heterotrimeric G protein beta subunit in Arabidopsis

Heterotrimeric G protein complexes are conserved from plants to mammals, but the complexity of each system varies. Arabidopsis thaliana contains one Gα, one Gβ (AGB1), and at least three Gγ subunits, allowing it to form three versions of the heterotrimer. This plant model is ideal for genetic studies because mammalian systems contain hundreds of unique heterotrimers. The activation of these complexes promotes interactions between both the Gα subunit and the Gβγ dimer with enzymes and scaffolds to propagate signaling to the cytoplasm. However, although effectors of Gα and Gβ are known in mammals, no Gβ effectors were previously known in plants. Toward identifying AGB1 effectors, we genetically screened for dominant mutations that suppress Gβ-null mutant (agb1-2) phenotypes. We found that overexpression of acireductone dioxygenase 1 (ARD1) suppresses the 2-day-old etiolated phenotype of agb1-2. ARD1 is homologous to prokaryotic and eukaryotic ARD proteins; one function of ARDs is to operate in the methionine salvage pathway. We show here that ARD1 is an active metalloenzyme, and AGB1 and ARD1 both control embryonic hypocotyl length by modulating cell division; they also may contribute to the production of ethylene, a product of the methionine salvage pathway. ARD1 physically interacts with AGB1, and ARD enzymatic activity is stimulated by AGB1 in vitro. The binding interface on AGB1 was deduced using a comparative evolutionary approach and tested using recombinant AGB1 mutants. A possible mechanism for AGB1 activation of ARD1 activity was tested using directed mutations in a loop near the substrate-binding site.     

Causes and consequences of variability in peptide mating pheromones of ascomycete fungi

The reproductive genes of fungi, like those of many other organisms, are thought to diversify rapidly. This phenomenon could be associated with the formation of reproductive barriers and speciation. Ascomycetes produce two classes of mating type-specific peptide pheromones. These are required for recognition between the mating types of heterothallic species. Little is known regarding the diversity or the extent of species specificity in pheromone peptides among these fungi. We compared the putative protein-coding DNA sequences of the 2 pheromone classes from 70 species of Ascomycetes. The data set included previously described pheromones and putative pheromones identified from genomic sequences. In addition, pheromone genes from 12 Fusarium species in the Gibberella fujikuroi complex were amplified and sequenced. Pheromones were largely conserved among species in this complex and, therefore, cannot alone account for the reproductive barriers observed between these species. In contrast, pheromone peptides were highly diverse among many other Ascomycetes, with evidence for both positive diversifying selection and relaxed selective constraint. Repeats of the α-factor-like pheromone, which occur in tandem arrays of variable copy number, were found to be conserved through purifying selection and not concerted evolution. This implies that sequence specificity may be important for pheromone reception and that interspecific differences may indeed be associated with functional divergence. Our findings also suggest that frequent duplication and loss causes the tandem repeats to experience "birth-and-death" evolution, which could in fact facilitate interspecific divergence of pheromone peptide sequences.     

Recent epidemiologic trends of diabetes mellitus among status Aboriginal adults

Background:

Little is known about longitudinal trends in diabetes mellitus among Aboriginal people in Canada. We compared the incidence and prevalence of diabetes, and its impact on mortality, among status Aboriginal adults and adults in the general population between 1995 and 2007.

Methods:

We examined de-identified data from Alberta Health and Wellness administrative databases for status Aboriginal people (First Nations and Inuit people with treaty status) and members of the general public aged 20 years and older who received a diagnosis of diabetes mellitus from Apr. 1, 1995, to Mar. 31, 2007. We calculated the incidence and prevalence of diabetes and mortality rate ratios by sex and ethnicity in 2007. We examined the average relative changes per year for longitudinal trends.

Results:

The average relative change per year in the prevalence of diabetes showed a smaller increase over time in the Aboriginal population than in the general population (2.39 v. 4.09, p < 0.001). A similar finding was observed for the incidence of diabetes. In the Aboriginal population, we found that the increase in the average relative change per year was greater among men than among women (3.13 v. 1.88 for prevalence, p < 0.001; 2.60 v. 0.02 for incidence, p = 0.001). Mortality among people with diabetes decreased over time to a similar extent in both populations. Among people without diabetes, mortality decreased in the general population but was unchanged in the Aboriginal population (−1.92 v. 0.11, p = 0.04). Overall, mortality was higher in the Aboriginal population than in the general population regardless of diabetes status.

Interpretation:

The increases in the incidence and prevalence of diabetes over the study period appeared to be slower in the status Aboriginal population than in the general population in Alberta, although the overall rates were higher in the Aboriginal population. Mortality decreased among people with diabetes in both populations but was higher overall in the Aboriginal population regardless of diabetes status.The health of Aboriginal people in Canada is generally poorer than their non-Aboriginal counterparts, and diabetes mellitus is a significant contributor.^1,2 Studies have shown that type 2 diabetes and its complications occur at rates two to five times higher in Canada’s Aboriginal population than in the general population.^3–7 In response, diverse diabetes programs have materialized, including various community-based prevention and screening projects.^8–10 The federally funded Aboriginal Diabetes Initiative was created to emphasize health promotion and diabetes prevention.¹¹ In addition, numerous Aboriginal communities have established their own diabetes and health programs.¹²Accurate diabetes surveillance data are essential for governments and health care organizations to plan health care delivery and translate knowledge into policy and funding decisions. However, research into the longitudinal trends of diabetes in Aboriginal populations is scarce. For the most part, data have come from small, community-based studies and self-reported surveys. Population-based studies of primary data are few and have been conducted only for limited periods. Even less is known about outcomes, mortality in particular, among Aboriginal individuals with diabetes.The use of administrative data is becoming more common for tracking diabetes in Canada.¹³ The National Diabetes Surveillance System uses administrative health data to document the burden of the disease, but it has little information on Aboriginal people. Dyck and colleagues recently used the methodology of the National Diabetes Surveillance System to examine the incidence and prevalence of diabetes among Aboriginal people in the province of Saskatchewan,¹⁴ and similar analyses were conducted in Manitoba and Ontario.^15,16As part of the Alberta Diabetes Surveillance System, we conducted this study to compare the incidence and prevalence of diabetes among people 20 years and older in the status Aboriginal population (First Nations and Inuit people with treaty status) and the general population in the province of Alberta between 1995 and 2007. We also compared trends in mortality in the two populations among people with and without diabetes.