ABSTRACT
Survivin, a recently identified inhibitor of apoptosis protein (IAP), is expressed in diverse embryonic tissues and in various human cancers. We have investigated the quantitative expression of survivin mRNA by a sensitive TaqMan-based RT-PCR assay in tissue samples from 94 patients with soft tissue sarcomas (STS). Survivin transcript levels were measured and normalized to GAPDH transcripts. By using a multivariate Cox regression analysis, we found an inverse correlation between the level of survivin mRNA (ratio >2 zmol survivin/amol GAPDH) and the rate of overall survival (p = 0.009, RR = 2.7). Survivin transcript variants as detected by qualitative RT-PCR analysis were revealed in 36 of 56 STS patients (64%). Only survivin DeltaEx3 and/or full-length survivin variants but not survivin 2B were identified. Our results suggest that a higher level of survivin mRNA is an independent predictor of survival for STS patients.
Subject(s)
Chromosomal Proteins, Non-Histone/biosynthesis , Microtubule-Associated Proteins , RNA, Messenger/metabolism , Sarcoma/diagnosis , Sarcoma/metabolism , Soft Tissue Neoplasms/diagnosis , Soft Tissue Neoplasms/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , DNA, Complementary/metabolism , Female , Humans , Inhibitor of Apoptosis Proteins , Male , Middle Aged , Multivariate Analysis , Neoplasm Proteins , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma/mortality , Soft Tissue Neoplasms/mortality , Survivin , Time FactorsABSTRACT
Following in vitro translation of the 22 kDa peroxisomal membrane protein (Pmp22p), gel filtration analysis of the post-ribosomal supernatant revealed that Pmp22p forms two complexes. Complex I is of high molecular weight, results in a crosslinking product of 80 kDa, and by co-immunoprecipitation with anti-TCP1 antibody was identified as TRiC. In complex II Pmp22p was crosslinked to a yet unknown polypeptide of 40 kDa (P40). This complex exhibited much higher efficiency to insert Pmp22p into the peroxisomal membrane compared to complex I. In a model we suggest that newly synthesized Pmp22p is first bound to TRiC before being transferred to P40 which may function as a cytosolic Pmp22p receptor.
Subject(s)
Intracellular Membranes/metabolism , Intracellular Signaling Peptides and Proteins , Membrane Proteins/metabolism , Microbodies/metabolism , Microtubule-Associated Proteins , Animals , Antibodies/immunology , Chaperonin Containing TCP-1 , Chaperonins/immunology , Chromatography, Gel , Cytosol/chemistry , Cytosol/metabolism , Electrophoresis, Polyacrylamide Gel , Liver/metabolism , Male , Mitochondria, Liver/metabolism , Nuclear Proteins/metabolism , Precipitin Tests , Protein Biosynthesis , Rats , Rats, Wistar , Reticulocytes/metabolism , Ubiquitin-Protein Ligases , t-Complex Genome RegionSubject(s)
Intracellular Membranes/metabolism , Membrane Proteins/metabolism , Microbodies/metabolism , Adenosine Triphosphate/metabolism , Animals , Biological Transport , Cell Compartmentation , Cloning, Molecular , Liver/ultrastructure , Peroxisomal Biogenesis Factor 2 , Rats , Structure-Activity Relationship , Sulfhydryl Compounds/chemistry , Temperature , Time FactorsABSTRACT
The membrane insertion of the 22-kD integral peroxisomal membrane protein (PMP 22) was studied in a system in which peroxisomes isolated from rat liver were incubated with the [35S]methionine-labeled in vitro translation product of PMP 22 mRNA. Membrane insertion of PMP 22 was demonstrated by protease treatment of peroxisomes in the absence and presence of detergent. Approximately 35% of total in vitro translated PMP 22 became protease resistant after a 1-h incubation at 26 degrees C. Import was dependent on time and temperature, did not require ATP or GTP and was not inhibited by N-ethylmaleimide treatment of neither the soluble components of the translation mixture nor of the isolated peroxisomes. In contrast to these results it was recently shown that the import of the peroxisomal marker, firefly luciferase, into peroxisomes of permeabilized cells was dependent on ATP hydrolysis and was blocked by N-ethylmaleimide pretreatment of the cytosol-depleted cells (Rapp et al., 1993; Wendland and Subramani, 1993). Therefore, the present data suggest that insertion of PMP 22 into the peroxisomal membrane and translocation of firefly luciferase into peroxisomes follow distinct mechanisms. At low temperature binding of PMP 22 to the peroxisomal membrane was not influenced whereas insertion was strongly inhibited. Pretreatment of peroxisomes with subtilisin reduced binding to a low level and completely abolished insertion. Therefore it is suggested that binding is prerequisite to insertion and that insertion may be mediated by a proteinaceous receptor.
Subject(s)
Liver/metabolism , Membrane Proteins/metabolism , Microbodies/metabolism , Animals , Cloning, Molecular , Coleoptera , Endopeptidases/pharmacology , Ethylmaleimide/pharmacology , Intracellular Membranes/metabolism , Kinetics , Luciferases/biosynthesis , Luciferases/metabolism , Membrane Proteins/biosynthesis , Methionine/metabolism , Microbodies/drug effects , Protein Biosynthesis , RNA, Messenger/metabolism , Rabbits , Rats , Recombinant Proteins/metabolism , Reticulocytes/metabolism , Sulfur RadioisotopesABSTRACT
In order to study the membrane topology and the possible function of the rat liver 22 kDa integral peroxisomal membrane protein (PMP 22) at a molecular level, we have cloned PMP 22 from a lambda gt11 expression library and sequenced its cDNA. Hydropathy analysis of the deduced primary structure indicates 4 putative transmembrane segments. The accessibility to exogenous aminopeptidase of PMP 22 in intact peroxisomes suggests that the N-terminus faces the cytosol. A model of the topology of PMP 22 in the peroxisomal membrane is discussed. Homology studies revealed a striking similarity with the Mpv 17 gene product. Lack of this membrane protein causes nephrotic syndrome in mice.
