ABSTRACT
Approximately 340 leucocyte plasma membrane proteins have been characterised by the eight Human Leucocyte Differentiation Antigen workshops held between 1982 and 2004, based primarily on their reactivity with monoclonal antibodies. The human genome is predicted to encode approximately 34,000 cDNA transcripts, of which between 15% and 20% are predicted to contain one or more transmembrane helices. We have used SDS-PAGE separation coupled with mass spectrometry-based peptide mass tag identification to identify novel plasma membrane proteins in microsome preparations prepared from mononuclear cells obtained from human peripheral blood. A total of 361 distinct proteins were identified in a single preparation, including 37 known leucocyte plasma membrane proteins, 27 potential novel plasma membrane proteins whose expression on PBMC is poorly characterised, and 51 other proteins for which the subcellular location could not be determined. Expression analysis using cDNA panels indicates that several of these novel plasma membrane proteins are differentially expressed in lymphocyte subsets. These results show that previously unidentified lymphocyte plasma membrane proteins can be identified using this approach.
Subject(s)
Electrophoresis, Polyacrylamide Gel , Leukocytes, Mononuclear/chemistry , Mass Spectrometry , Membrane Proteins/analysis , Microsomes/chemistry , Cell Membrane/chemistry , DNA, Complementary/genetics , Humans , Membrane Proteins/geneticsABSTRACT
Cells respond to a wide variety of stresses through the transcriptional activation of genes that harbour stress elements within their promoters. While many of these elements are shared by genes encoding proteins representative of all subcellular compartments, cells can also respond to stresses that are specific to individual organelles, such as the endoplasmic reticulum un folded protein response. Here we report on the discovery and characterization of a mitochondrial stress response in mammalian cells. We find that the accumulation of unfolded protein within the mitochondrial matrix results in the transcriptional upregulation of nuclear genes encoding mitochondrial stress proteins such as chaperonin 60, chaperonin 10, mtDnaJ and ClpP, but not those encoding stress proteins of the endoplasmic reticulum. Analysis of the chaperonin 60/10 bidirectional promoter identified a CHOP element as the mitochondrial stress response element. Dominant-negative mutant forms of CHOP and overexpression of CHOP revealed that this transcription factor, in association with C/EBPbeta, regulates expression of mitochondrial stress genes in response to the accumulation of unfolded proteins.
Subject(s)
Heat-Shock Proteins/biosynthesis , Mitochondria/physiology , Stress, Physiological/physiopathology , Adenosine Triphosphatases/biosynthesis , Adenosine Triphosphatases/genetics , Animals , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/physiology , COS Cells , Cell Nucleus/metabolism , Chaperonin 10/biosynthesis , Chaperonin 10/genetics , Chaperonin 60/biosynthesis , Chaperonin 60/genetics , Chlorocebus aethiops , Endopeptidase Clp , Gene Expression Regulation , Genes, Dominant , HSP40 Heat-Shock Proteins , Heat-Shock Proteins/genetics , Ornithine Carbamoyltransferase/chemistry , Ornithine Carbamoyltransferase/genetics , Ornithine Carbamoyltransferase/metabolism , Promoter Regions, Genetic , Protein Folding , Protein Processing, Post-Translational , Protein Transport , Recombinant Fusion Proteins/physiology , Regulatory Sequences, Nucleic Acid , Sequence Deletion , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics , Signal Transduction , Stress, Physiological/genetics , Transcription Factor CHOP , Transcription Factors/genetics , Transcription Factors/physiology , Transcription, Genetic , TransfectionABSTRACT
Controlled production of the cytokine IL-2 plays a key role in the mammalian immune system. Expression from the gene is tightly regulated with no detectable expression in resting T cells and a strong induction following T cell activation. The IL-2 proximal promoter (+1 to -300) contains many well-defined transcriptional activation elements that respond to T cell stimulation. To determine the role of chromatin structure in the regulation of interleukin-2 gene transcription, nucleosome assembly across the IL-2 promoter region was examined using in vitro chromatin reconstitution assays. The IL-2 promoter assembles a nucleosome that is both translationally and rotationally positioned, spanning some of the major functional control elements. The binding of transcription factors to these elements, with the exception of the architectural protein HMGA1, was occluded by the presence of the nucleosome. Analysis of the chromatin architecture of the IL-2 gene in Jurkat T cells provided evidence for the presence of a similarly positioned nucleosome in vivo. The region encompassed by this nucleosome becomes remodeled following activation of Jurkat T cells. These observations suggest that the presence of a positioned nucleosome across the IL-2 proximal promoter may play an important role in maintaining an inactive gene in resting T cells and that remodeling of this nucleosome is important for gene activation.