“Stress Proteins”• Ubiquitin• Metallothioneins•HemeOxygenase• Heat Shock ProteinsNormal function:• “molecular chaperones” which maintain the homeostasis of the cell by facilitating the folding, assembly, and distribution of newly synthesized proteins;• Facilitate cross-membrane and intra-cellular transport;• Prevent anomalous conformation of some proteins (e.g., hormone receptors);• Immune system role – transport of peptides in/out of the cell1) ubiquitin activation by E1 (ubiquitin-activating enzyme) followed by ubiquitindelivery to E2 (ubiquitin-conjugating enzyme)2) complex formation by E2-Cys Ub, E3 (ubiquitin ligase) and the substrate 3) transfer of ubiquitins to the substrate lysine(s) to earmark the substrate with a polyubiquitin chain. 4) a polyubiquitylated substrate is released from the E3. Proteasomes recognize the polyubiquitin chain as a signal to de-ubiquitylate and destroy the substrate. 5) the proteasome unfolds the substrate in ATP-dependent manner, removes the ubiquitin chain through a proteasome-associated ubiquitin hydrolase activity, and threads the unfolded protein into the proteasome chamber, where the protease active sites are located6) the ubiquitin molecules are recycled, and the peptides generated are used in major histocompatibility antigen presentation or degraded to amino acids that are recycled for new protein synthesis Nature Reviews Drug Discovery 5, 596-613 (July 2006)Ubiquitin–proteasome systemwww.med.unibs.itEnvironmental Health Perspectives 112(4), 2004Induction of HO-1 by cysplatin in rat kidney cellsHeme Oxygenase• Induced by metals and a number of xenobiotics• CO is produced: potent vasodilator• bilirubin and biliverdin are potent antioxidantsMetallothioneins• small (6-7 kDa), cysteine rich (30% of AA), metal binding proteins• located in cytoplasm, lysosomes, nucleus• major intracellular zinc binding proteins– zinc and copper homeostasis• highly inducible– Cd, Zn, Cu• sequestration of Cd– overwhelming induction in cells exposed to CdNormal function(a) The function in normal cells is probably the formation of a complex with Zn2+ and as an intracellular storage form of Zn2+, and perhaps other ions such as Cu2+(b) Metallothioneins also appear to act as free-radical scavengers(c) A clearly established role of metallothionein is its protective function against exposure to acute toxic doses of Cd2+ and otherheavy metallic cations in rodents or in cultured mammalian cellsMT or induced synthesis of MT (e.g. mRNA for MT) is protectiveMetallothioneinsRapid synthesis and degradation of MT mRNAThe response of cells to the need for metallothionein upon exposure to toxic metallic cations (and other inducers) is moderately rapid (see Figure).Treatment of rats or mice with Cd2+ results in a 3-to 5-fold increase in the concentration of mRNA for metallothionein (specifically metallothionein- I , MT- 1 ) by three hours and a peak of 12-fold in mice and a 7-fold increase in rats at 6 hours after treatment with Cd2+.The amount of MT-l mRNA had decreased to approximately 2-fold above control by 12 hours in mice and by 18 hours in rats. Lipopolysaccharide, which also induces the synthesis of metallothionein, also caused a peak increase at 6 hours alter treatment with a similar-fold increase in the MT-l mRNA to that induced by Cd2+. Toxicology and Applied Pharmacology 19: 1-10 (1993)0 100 200 300 400 5000102030405060708090100110120 Metallothionein producing cells Metallothionein deficient cellsProportional Survival (%)Cadmium Dose (nM)Metallothionein protection in vitroMetallothionein and Renal Toxicity• Cd absorbed into body and initially accumulates in liver• Cd-GSH complexes excreted into bile (subject to enterohepatic cycling)• MT-Cd complexes formed and slowly leak into systemic circulation• MT-Cd complexes then accumulate in kidney• MT is protective but has a threshold or protection limit– Protects through sequestration of Cd within cells– Also causes accumulation– Rapid lysosomal degradation of Cd-MT complex • Massive concentrations of Cd released --toxicity• response in cells exposed to elevated temperature/stress stimuli• highly conserved throughout evolution from Escherichia coli to human • a wide variety of biological (infection, inflammation), physical (radiation, hypoxia) and chemical (alcohols, metals) stressors can induce the response Function: major HSPs are molecular chaperones with an essential role in directing protein folding and assembly of polypeptides within the cellMechanism of induction: under stress (increased temperature/xenobiotics, etc.), the proportion of misfolded proteins (MFPs) suddenly increases Æ synthesys of HSPs to assist in protein refolding:• controlled primarily at the transcription level by a heat shock factor (HSF)• unstressed cells: HSF is present in the cytoplasm and the nucleus in a monomeric form that has no DNA binding activity through its interactions with HSPs• in response to stress, the monomeric HSFs Æ trimers and accumulate within the nucleuscombio.abo.fiHeat Shock Proteins (HSPs)Tolerance of extreme temperatureHsp104, Hsp110ClpB, ClpA, ClpX100 kDaMaintenance of steroid receptors and transcription factorsThe HspC group of Hspincluding Hsp90, Grp94HtpG, C62.590 kDaProtein folding and unfolding, provide thermotolerance to cell during heat stress. Also prevent protein folding during post-translational import into the mitochondria/chloroplast.The HspA group of Hspincluding Hsp71, Hsc70, Hsp72, Grp78 (BiP), Hsx70 found only in primatesDnaK70 kDaInvolved in protein folding after its post-translational import to the mitochondrion/chloroplastHsp60GroEL, 60kDa antigen60 kDaHsp40DnaJ40 kDaThe HspB group of Hsp. Ten members in mammals including Hsp27 or HspB1GrpE20-30 kDaHsp10GroES10 kDaFunctionEukaryotic proteinsProkaryoticproteinsApproximate molecular weight (kDa)Hsp27• Stabilizes damaged (e.g., denatured) proteins: actin cytoskeleton• Prevents apoptosis by blocking:• release of cytochrome c• activation of caspase 9• DAXX-Fas dimerization• Induces production of GSHHsp60• Chaperones: guide synthesis, transport and degradation of proteins• Highly abundant in all cells and conserved through evolution• Rapidly induced upon “stress” to cope with misfolded and aggregated proteins• Increases in expression are frequently related to an increase in inflammatory cytokinesHsp70• Chaperones: guide synthesis, transport and
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