RESUMO
The assembly of individual mammalian proteasome subunits into catalytically active 20S proteasome is not well understood. Herein, we report the identification and characterization of human and mouse homologues of the yeast proteasome maturating factor Ump1p. We delineate the region of hUMP1 implicated in the specific interaction with proteasome precursors and show that hUMP1 protein is absent from the mature form of the 20S proteasome. We also show that the transcript level of mammalian UMP1 is increased after IFN-gamma treatment and that mammalian UMP1 is functionally related to but not interchangeable with its yeast homologue.
Assuntos
Cisteína Endopeptidases/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multienzimáticos/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Clonagem Molecular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
IFN-gamma induces a number of cellular programs functional in innate and adaptive resistance to infectious pathogens. It has recently become clear that the complete cellular response to IFN-gamma is extraordinarily complex, with >500 genes (i.e., approximately 0.5% of the genome) activated. We made suppression-subtractive hybridization differential libraries from IFN-gamma-stimulated primary mouse embryonic fibroblasts and from a mouse macrophage cell line, ANA-1, in each case with reference to unstimulated cells. Of approximately 250 clones sequenced at random from the two libraries, >35% were representatives of one or the other of two small unrelated families of GTPases, the 65-kDa and 47-kDa families. These families dominate the IFN-gamma-induced response in both cell types. We report here the full-length sequences of one new 65-kDa and two new 47-kDa family members. The 65-kDa family members are under transcriptional control of IRF-1, whereas the 47-kDa family members are inducible in embryonic fibroblasts from IRF-1(-/-) mice. Members of both GTPase families are strongly up-regulated in livers of wild-type mice infected with the pathogenic bacterium, Listeria monocytogenes, but not in IFN-gammaR(0/0) mice. These GTPases appear to be dedicated to the IFN-gamma response, since resting levels are negligible and since neither family shows any significant relationship to any other described family of GTPases. Understanding the role of these GTPases in IFN-gamma-mediated resistance against pathogens is the task for the future.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fibroblastos/enzimologia , GTP Fosfo-Hidrolases/classificação , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Macrófagos/enzimologia , Fosfoproteínas/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Evolução Molecular , Fibroblastos/efeitos dos fármacos , GTP Fosfo-Hidrolases/fisiologia , Biblioteca Gênica , Fator Regulador 1 de Interferon , Listeriose/imunologia , Fígado/enzimologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Peso Molecular , Fosfoproteínas/genética , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Técnica de Subtração , Transcrição Gênica , Receptor de Interferon gamaRESUMO
Interferons are cytokines that play a complex and central role in the resistance of mammalian hosts to pathogens. Type I interferon (IFN-alpha and IFN-beta) is secreted by virus-infected cells. Immune, type II, or gamma-interferon (IFN-gamma) is secreted by thymus-derived (T) cells under certain conditions of activation and by natural killer (NK) cells. Although originally defined as an agent with direct antiviral activity, the properties of IFN-gamma include regulation of several aspects of the immune response, stimulation of bactericidal activity of phagocytes, stimulation of antigen presentation through class I and class II major histocompatibility complex (MHC) molecules, orchestration of leukocyte-endothelium interactions, effects on cell proliferation and apoptosis, as well as the stimulation and repression of a variety of genes whose functional significance remains obscure. The implementation of such a variety of effects by a single cytokine is achieved by complex patterns of cell-specific gene regulation: Several IFN-gamma-regulated genes are themselves components of transcription factors. The IFN-gamma response is itself regulated by interaction with responses to other cytokines including IFN-alpha/beta, TNF-alpha, and IL-4. Over 200 genes are now known to be regulated by IFN-gamma and they are listed in a World Wide Web document that accompanies this review. However, much of the cellular response to IFN-gamma can be described in terms of a set of integrated molecular programs underlying well-defined physiological systems, for example the induction of efficient antigen processing for MHC-mediated antigen presentation, which play clearly defined roles in pathogen resistance. A promising approach to the complexity of the IFN-gamma response is to extend the analysis of the less understood IFN-gamma-regulated genes in terms of molecular programs functional in pathogen resistance.
