Study of the effects of proteasome inhibitors on ribosomal protein S19 (RPS19) mutants, identified in patients with Diamond-Blackfan anemia.

BACKGROUND: Mutations in the ribosomal protein S19 gene (RPS19) have been found in 25% of patients with Diamond-Blackfan anemia, a rare syndrome of congenital bone marrow failure characterized by erythroblastopenia and various malformations. Mechanistic understanding of the role of RPS19 in normal erythropoiesis and in the Diamond-Blackfan anemia defect is still poor. However, defective ribosome biogenesis and, in particular, impaired 18S ribosomal RNA maturation have been documented in association with various identified RPS19 mutations. Recently, new genes, all encoding ribosomal proteins, have been found to be mutated in Diamond-Blackfan anemia, adding further support to the concept that ribosome biogenesis plays an important role in regulating erythropoiesis. We previously showed variability in the levels of expression and subcellular localization of a subset of RPS19 mutant proteins. DESIGN AND METHODS: To define the mechanistic basis for this variability better, we studied a large number of mutant proteins and characterized both RPS19 expression level using a specific antibody against RPS19 and RPS19 subcellular localization after transfection of Cos-7 cells with various green fluorescent protein-RPS19 mutants. To investigate the role of the proteasome in RPS19 degradation, we examined the effect of various proteasome inhibitors, namely lactacystin, MG132, and bortezomib on RPS19 expression and subcellular localization RESULTS: We found two distinct classes of RPS19 protein defects in Diamond-Blackfan anemia based on the stability of the mutant proteins: (i) slightly decreased to normal levels of expression and normal nucleolar localization and (ii) markedly deficient expression and failure to localize to the nucleolus. All the proteasome inhibitors tested were able to restore the expression levels and normal subcellular localization of several unstable mutant proteins. CONCLUSIONS: Our findings demonstrate an important role for the proteasomal degradation pathway in regulating the expression levels and nucleolar localization of certain mutant RPS19 proteins in Diamond-Blackfan anemia.

Role of uncoupling protein UCP2 in cell-mediated immunity: how macrophage-mediated insulitis is accelerated in a model of autoimmune diabetes.

Infiltration of inflammatory cells into pancreatic islets of Langerhans and selective destruction of insulin-secreting beta-cells are characteristics of type 1 diabetes. Uncoupling protein 2 (UCP2) is a mitochondrial protein expressed in immune cells. UCP2 controls macrophage activation by modulating the production of mitochondrial reactive oxygen species (ROS) and MAPK signaling. We investigated the role of UCP2 on immune cell activity in type 1 diabetes in Ucp2-deficient mice. Using the model of multiple low-dose streptozotocin (STZ)-induced diabetes, we found that autoimmune diabetes was strongly accelerated in Ucp2-KO mice, compared with Ucp2-WT mice with increased intraislet lymphocytic infiltration. Macrophages from STZ-treated Ucp2-KO mice had increased IL-1beta and nitric oxide (NO) production, compared with WT macrophages. Moreover, more macrophages were recruited in islets of STZ-treated Ucp2-KO mice, compared with Ucp2-WT mice. This finding also was accompanied by increased NO/ROS-induced damage. Altogether, our data show that inflammation is stronger in Ucp2-KO mice and islets, leading to the exacerbated disease in these mice. Our results highlight the mitochondrial protein UCP2 as a new player in autoimmune diabetes.

Avian UCP: the killjoy in the evolution of the mitochondrial uncoupling proteins.

The understanding of mitochondrial functioning is of prime importance since it combines the production of energy as adenosine triphosphate (ATP) with an efficient chain of redox reactions, but also with the unavoidable production of reactive oxygen species (ROS) involved in aging. Mitochondrial respiration may be uncoupled from ATP synthesis by a proton leak induced by the thermogenic uncoupling protein 1 (UCP1). Mild uncoupling activity, as proposed for UCP2, UCP3, and avian UCP could theoretically control ROS production, but the nature of their transport activities is far from being definitively understood. The recent discovery of a UCP1 gene in fish has balanced the evolutionary view of uncoupling protein history. The thermogenic proton transport of mammalian UCP1 seems now to be a late evolutionary characteristic and the hypothesis that ancestral UCPs may carry other substrates is tempting. Using in silico genome analyses among taxa and a biochemical approach, we present a detailed phylogenetic analysis of UCPs and investigate whether avian UCP is a good candidate for pleiotropic mitochondrial activities, knowing that only one UCP has been characterized in the avian genome, unlike all other vertebrates. We show, here, that the avian class seems to be the only vertebrate lineage lacking two of the UCP1/2/3 homologues present in fish and mammals. We suggest, based on phylogenetic evidence and synteny of the UCP genes, that birds have lost UCP1 and UCP2. The phylogeny also supports the history of two rounds of duplication during vertebrate evolution. The avian uncoupling protein then represents a unique opportunity to explore how UCPs' activities are controlled, but also to understand why birds exhibit such a particular relationship between high metabolism and slow rate of aging.

Mitochondria contribute to LPS-induced MAPK activation via uncoupling protein UCP2 in macrophages.

The mitochondrion is a major organelle contributing to energy metabolism but also a main site of ROS (reactive oxygen species) production. LPS (lipopolysaccharide)-induced ROS signalling is a critical event in macrophage activation. In the present paper we report that part of LPS-mediated ROS signalling comes from mitochondria inside a signal amplification loop that enhances MAPK (mitogen-activated protein kinase) activation. More precisely, we have identified the inner mitochondrial membrane UCP2 (uncoupling protein 2) as a physiological brake on ROS signalling. Stimulation of murine bone marrow-derived macrophages by LPS quickly down-regulated UCP2 through the JNK (c-Jun N-terminal kinase) and p38 pathways. UCP2 down-regulation was shown to be necessary to increase mitochondrial ROS production in order to potentiate MAPK activation. Consistent with this, UCP2-deficient macrophages exhibit an enhanced inflammatory state characterized by increased nitric oxide production and elevated migration ability. Additionally, we found that the absence of UCP2 renders macrophages more resistant to nitric oxide-induced apoptosis.

The uncoupling protein 2 modulates the cytokine balance in innate immunity.

The uncoupling protein 2 (UCP2) is located in the inner mitochondrial membrane and downregulates the production of reactive oxygen species (ROS). Recent data suggested a role for UCP2 in the immune response. We analyzed further this hypothesis during acute Listeria monocytogenes infection in mice. Death of infected Ucp2(-/-) mice was delayed in comparison with Ucp2(+/+), suggesting a role of UCP2 in the early step of the immune response. In vitro, the higher resistance of Ucp2(-/-) mice was not associated with a better control of bacterial growth by macrophages. In vivo, a significant increase of recruited phagocytes was observed in the spleen of Ucp2(-/-) mice. This was associated with a higher level of ROS in the spleen. Upregulation of pro-inflammatory cytokines IFNgamma, IL6, and IL1beta and of the chemokine MCP1 was observed in Ucp2(-/-) mice 4 days after infection, preceded by a decrease of the anti-inflammatory cytokine IL10 production. Present data highlight that, in an acute model of infection, UCP2 modulates innate immunity, via the modulation of ROS production, cytokine and chemokine production and consequently phagocyte recruitment.

UCP2, UCP3, avUCP, what do they do when proton transport is not stimulated? Possible relevance to pyruvate and glutamine metabolism.

Uncoupling proteins (UCPs) are specialized members of the mitochondrial transporter family. They allow passive proton transport through the mitochondrial inner membrane. This activity leads to uncoupling of mitochondrial respiration and to energy waste, which is well documented with UCP1 in brown adipose tissue. The uncoupling activity of the new UCPs (discovered after 1997), such as UCP2 and UCP3 in mammals or avUCP in birds, is more difficult to characterize. However, extensive data support the idea that the new UCPs are involved in the control of reactive oxygen species (ROS) generation. This fits with the hypothesis that mild uncoupling caused by the UCPs prevents ROS production. Activators and inhibitors regulate the proton transport activity of the UCPs. In the absence of activators of proton transport, the UCP allows the permeation of other ions. We suggest that this activity has physiological significance and, for example, UCP3 expressed in glycolytic muscle fibres may be a passive pyruvate transporter ensuring equilibrium between glycolysis and oxidative phosphorylation. Induction of UCP2 expression by glutamine strengthens the proposal that new UCPs could act to determine the choice of mitochondrial substrate. This would obviously have an impact on mitochondrial bioenergetics and ROS production.