Our group was among the first to demonstrate TRAP1involvement in stress-adaptive response of cancer cells: highlevels of both TRAP1 mRNA and protein were found in Saos-2osteosarcoma cells chronically adapted to mild oxidative conditions(). Even more interestingly, weidentified TRAP1 as a key target in the previously hypothesizedcorrelations between resistance to antitumor agents and adaptationto oxidative stress (OS), since very high levels of this proteinwere analogously found in tumor cells resistant to 5-fluorouraciland to platin derivatives. However, the most striking data camefrom the observation that TRAP1 interference, as well as the use ofdominant negative mutants of TRAP1, sensitized OS/chemoresistantcells to cell death inducers, thus supporting the hypothesis ofcommon mechanisms shared by chemoresistance and adaptation to OSand providing the first evidence that TRAP1 is an important playerin the development and the maintenance of these phenotypes. Indeed,TRAP1 hyperexpressing cells show a decreased cleavage of theapoptotic markers Caspase 3 and PARP, and increased levels of thescavenging tripeptide GSH. Hence, TRAP1 may be considered areliable tool to investigate the correlations between oxidativestress, resistance to apoptosis and chemoresistance (,).
A role of TRAP1 in the protection from apoptosis andits consequent involvement in the onset and maintenance of tumorphenotypes was firstly and elegantly described by Kang (). These authors discovered thatonly tumor cells organize a mitochondrial chaperone network, whichinvolves HSP90, its homolog TRAP1 and the immunophilin cyclophilinD (CypD) in a physical complex that regulates permeabilitytransition pore (PTP) opening, maintaining mitochondrialhomeostasis and antagonizing the function of CypD in permeabilitytransition. Considering the high ‘druggability’ of HSP90 ATPasepocket, Kang and colleagues developed a mitochondria-directedvariant of 17-AAG carrying the (calledShepherdin) that efficiently accumulates inside the mitochondria,binds mitochondrial TRAP1 and HSP90, and inhibits their chaperoneactivity via an ATP competition mechanism, thus resulting inCypD-mediated cell death. These observations labelled TRAP1 as anessential controller of mitochondrial homeostasis in tumor cells,conferring them resistance to apoptosis and a survival advantageover the normal counterpart ().The cytoprotective effect of TRAP1 was further investigated by ourgroup through the identification and functional characterization ofTRAP1 interaction with the novel mitochondrial isoform of Sorcin,this providing the first demonstration of a new antiapoptoticcomplex ().
Protein Folding and Processing in the ER: Various changes occur to proteins in the ER. Chaperones and Folding: Polypeptides must assume the correct folding pattern in order to function properly. The correct folding of a protein is mediated by chaperones (they also are proteins--chaperones are abundant in the ER lumen). A completed polypeptide will assume the correct folding pattern spontaneously, however before translation is complete, it could assume an incorrect formation or it could aggregate with other partially made polypeptides. To prevent this, chaperones in the ER (and cytosol) bind to the nascent polypeptide and keep it from interacting with anything until the polypeptide is completely synthesized. (Chaperones bind to polypeptides destined for mitochondria then release them as they pass through the mitochondrial membranes. Chaperones on the inside of mitochondria bind until these polypeptides have completely entered.)
The diagram at the left summarizes the flow of proteins between the compartments of the endomembrne system. In the initial section of this unit we will consider the transfer of proteins from ER to golgi (red arrow) and the reverse (retrograde) transfer of receptors and proteins destined for retention in the ER compartment (blue arrow). The non highlighted portion of the figure shows the flow of proteins in the secretory, lysosomal and endocytotic pathways that will be considered later. Click on image to enlarge.
AB - Background - Heat shock proteins (HSPs) are well known for their ability to "protect" the structure and function of native macromolecules, particularly as they traffic across membranes. Considering the role of key mitochondrial proteins in apoptosis and the known antiapoptotic effects of HSP27 and HSP72, we postulated that HSP60, primarily a mitochondrial protein, also exerts an antiapoptotic effect. Methods and Results - To test this hypothesis, we used an antisense phosphorothioate oligonucleotide to effect a 50% reduction in the levels of HSP60 in cardiac myocytes, a cell type that has abundant mitochondria. The induced decrease in HSP60 precipitated apoptosis, as manifested by the release of cytochrome c, activation of caspase 3, and induction of DNA fragmentation. Antisense treatment was associated with an increase in bax and a decrease in bcl-2 secondary to increased synthesis of bax and degradation of bcl-2. A control oligonucleotide had no effect on these measurements. We further demonstrated that cytosolic HSP60 forms a macromolecular complex with bax and bak in vitro suggesting that complex formation with HSP60 may block the ability of bax and bak to effect apoptosis in vivo. Lastly, we show that as cytosolic (nonmitochondrial) HSP60 decreases, a small unbound fraction of bax appears and that the amount of bax associated with the mitochondria and cell membranes increases. Conclusions - These results support a key antiapoptotic role for cytosolic HSP60. To our knowledge, this is the first report suggesting that interactions of HSP60 with bax and/or bak regulate apoptosis.
According to the , 1070 proteins have been detected in the human mitochondria. Thus, since 13 of them are from mitochondrial origin, we conclude that at least 98.7% of the mitochondrial proteins are imported from the cytosol.
To prevent this, chaperones in the ER (and cytosol) bind to the nascent polypeptide and keep it from interacting with anything until the polypeptide is completely synthesized.
Some proteins are retained in the ER (for example, the enzymes that make the oligosaccharides that are added to proteins) These proteins carry an ER retention signal (KDEL or MDEL sequence) at their carboxyl ends. See Table 14-3. Even if they get out of the ER into the cisternae of the Golgi, their ER targeting signal gets them sorted into vesicles that bring them back to the ER. This cis-ward movement of vesicles is called movement.
Although twenty years have passed since TRAP1/HSP75was firstly identified, only during recent years some light hasbeen shed on its molecular functions. The cloning of tumor necrosisfactor receptor-associated protein 1 (TRAP1) as a type I tumornecrosis factor receptor-associated protein, and the identificationof HSP75 as a retinoblastoma protein (Rb)-binding protein, wereindependently performed by two different groups, and it wasimmediately clear that they had identified the same protein(,). TRAP1 belongs to the HSP90 chaperonefamily (), sharing 26% identityand 45% similarity with cytosolic HSP90 (). Bioinformatic analysis and microscopicobservations suggest that TRAP1 is mostly localized to mitochondriaand is targeted to the organelle by its N-terminal presequence(). Interestingly, the ATPaseactivity of TRAP1 is inhibited by both geldanamycin and radicicol,which have been shown to block specifically HSP90 function;however, experiments showed that TRAP1 does notbind and fold HSP90 client proteins, suggesting distinct functionalproperties. Quantitative immunogold electron microscopy andbiochemical analysis confirmed the mitochondrial distribution ofTRAP1 in rat tissues, but additionally revealed a number ofnon-mitochondrial locations, including nuclei ().
Disulfide bridges are found only in proteins that are to be secreted or are exterior membrane proteins, since the cytosol contains reducing agents that would break these S-S bonds.
Start by keeping in mind that proteins contained in the cisternae of the ER are separated from the cytosol by a membrane. If these proteins are to be moved, they must be moved as part of membrane vesicles, and any enzymes that act on the proteins must be contained in the vesicles or cisternae that contain the proteins.
The present study used pharmacological, biochemical, and behavioral methods to examine the role of protein synthesis in the hippocampus in memory processes of a passive avoidance learning in rats. Results indicated that corticotropin-releasing factor (CRF) significantly improved memory retention in rats. Both cycloheximide (CHX) and actinomycin-D (ACT-D) impaired memory at high doses. At doses of CHX and ACT-D that did not affect memory alone, they both antagonized the memory-enhancing effect of CRF. Biochemically, there were specific increases in the optical density of three protein bands in the cytosolic fraction of hippocampal cells in rats showing good memory. There were also marked increases in the optical density of two protein bands in the nucleus fraction of the same animals. Similar results were observed in animals injected with CRF. However, no significant protein alteration was observed in animals receiving stress. These results together suggest that there are new protein syntheses in the hippocampus that are specifically associated with passive avoidance learning in rats.