Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.25.1 (proteasome)
 28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mitochondrion depends upon the import of cytosolically synthesized preproteins for most of the proteins that comprise its structural elements and metabolic pathways. Here we have examined the influence of redox conditions on mitochondrial preprotein import and processing by mammalian mitochondria. Paraquat pretreatment of isolated mitochondria inhibited the subsequent import preornithine transcarbamylase (pOTC) in vitro. In intact cells oxidizing conditions led to decreased levels of mature OTC and accumulation of its preprotein. Implicating a mitochondrial import lesion, the fluorescence of pOTC-GFP (a protein in which the presequence of pOTC was fused to green fluorescent protein) transfected cells was decreased by paraquat treatment while cytosolic wild-type GFP remained largely unaffected. The accumulation of preproteins was enhanced by proteasome inhibitors. We observed that precursor proteins that failed to be imported, due to oxidizing conditions or an intrinsically slower import rate, are susceptible to degradation. Inhibition of the proteasome was also found to lead to higher levels of the translocase outer membrane protein 20 (Tom20) and to the perinuclear accumulation of mitochondria. These studies indicate that cellular redox conditions influence mitochondrial import, which, in turn, affects mitochondrial protein levels. A role for the proteasome in this process and in general mitochondrial function was also indicated.
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PMID:Oxidative stress inhibits the mitochondrial import of preproteins and leads to their degradation. 1116 10

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide-glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG-tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes.
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PMID:Plasmid vectors for proteomic analyses in Giardia: purification of virulence factors and analysis of the proteasome. 2261 Oct 20