Inclusion bodies as reversible protein reservoirs
1
TRENDS in Biochemical Sciences Vol.26 No.3 March 2001 http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. 151 News & Comment Journal Club Inclusion bodies (IBs) are major protein aggregates that arise owing to a deficiency of the cell’s protein folding machinery. This often occurs in recombinant bacteria where plasmid-encoded genes are being highly expressed. In this context, research into the prevention of IB formation or the re-forming of correctly folded protein from IBs is of great importance to the biotechnology industry. Understanding IBs is equally important for research into amyloid diseases (such as Alzheimer’s disease), which are caused by misfolding of proteins, resulting in their deposition in the cells as fibrils. Surprisingly, given the significance of IBs, they have generally been poorly investigated. However, a recent paper by Carrió et al. 1 is an exception. They have shown that IB production in vivo is, in fact, caused by an imbalance between protein aggregation and solubility. The authors used two plasmid-encoded proteins expressed in Escherichia coli to study IB production. One was a β-galactosidase fused to a viral capsid protein, and the other was phage 22 tail-spike protein (TSP). In both systems, they calculated the number of IBs formed and their volume, the amount of soluble versus insoluble protein, and the activity of the protein as an indicator of proper folding. These measurements were taken at hourly intervals before and after the addition of chloramphenicol, which arrests protein synthesis. In the β-galactosidase system, using SDS-PAGE and densitometry against known standards, they showed that the amount of insoluble material in the cell cultures sharply decreased upon addition of chloramphenicol, with a concomitant increase in the amount of soluble material. A combination of microscopy and digital imaging also showed a reduction in the number and volume of IBs in the cells. Correct re-folding of the protein was confirmed by an increase in the β-galactosidase activity in the cultures following termination of protein synthesis. The same trend was found with the expression of TSP. Carrió et al. also demonstrated in vitro re-folding of IB proteins by incubating purified IBs from the two strains with cell extracts from plasmid-free BL26 cells, with the result being a recovery of enzymatic activity and soluble peptide in both systems. The authors concluded that IBs are the result of an imbalance in the equilibrium between protein precipitation and re-folding and solubility in the cell. By switching off protein production, the protein folding machinery can catch up, thus redressing the balance. The authors therefore see IBs not as a dead-end product caused by inefficiency of the bacterial protein folding machinery, but rather as a reversible reservoir of protein awaiting correct folding when conditions permit. The significance of the result is already apparent: IB solubility in vitro could provide the answer to the problem of IB formation in biotechnology and, from a clinical viewpoint, it raises possibilities for new therapeutic approaches to amyloid diseases. 1 Carrió, M.M. et al. (2000) Fine architecture of bacterial inclusion bodies. FEBS Lett. 471, 7–11 James Pearce [email protected] Inclusion bodies as reversible protein reservoirs Repair of DNA damage by the versatile nucleotide excision repair (NER) pathway proceeds much faster in the transcribed strand of an active gene than it does in the non-transcribed strand or in the global genome. This transcription-coupled repair (TCR) was first described almost 15 years ago but, despite its obvious importance for maintenance of genome integrity, little has been learned about the molecular mechanisms that make such preferential repair possible. New results from the Brouwer laboratory 1 now suggest that TCR works better when the efficiency of transcriptional elongation is relaxed. Two genes, Cockayne’s Syndrome A (CSA) and B (CSB), encode proteins that are involved in this process. The yeast homologue of CSB is RAD26 and, like mammalian CS cells, rad26 cells show a markedly slower repair of the transcribed strand of an active gene than do normal cells. CSB and Rad26 are DNA-dependent ATPases often thought of as possible functional analogues of the bacterial transcription coupling repair factor (TCRF) protein, which interacts directly with the bacterial NER machinery and is capable of displacing a stalled RNA polymerase, thus enabling DNA repair. The new results 1 show that in an active yeast gene, where TCR was previously shown to be completely dependent on RAD26, fast preferential repair of the transcribed strand can actually take place in the absence of RAD26, provided the SPT4 gene is also deleted. Spt4 and its partner Spt5 form a complex [DRB sensitivity- inducing factor (DSIF) in mammalian cells] that assists transcription elongation and interacts with RNA polymerase II. Genetic interactions between SPT4 and NER factors indicate an even closer relationship between transcriptional elongation and NER than previously described. These data suggest that, in contrast to TCRF, Rad26 (and, thereby in all likelihood, CSB) is not involved in the TCR reaction per se, and point to RNA polymerase II elongation complexes as important determinants for the efficiency of TCR. Different models to explain the possible role of Rad26 in TCR could be suggested. However, one simple interpretation is that, in the presence of DNA damage, efficient transcriptional elongation becomes counter-productive. During such adversity, Rad26 and CSB might help to prevent the RNA polymerase II elongation complexes from ‘choking’ on the damage. When Spt4 is missing from cells, transcription elongation efficiency might already be reduced, thus eliminating the need for Rad26 and CSB. 1 Jansen, L.T. et al. (2000) Spt4 modulates Rad26 requirement in transcription-coupled nucleotide excision repair. EMBO J. 19, 6498–6507 Jesper Q. Svejstrup [email protected] Transcription-coupled DNA repair without the transcription-coupling repair factor
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Transcript of Inclusion bodies as reversible protein reservoirs
TRENDS in Biochemical Sciences Vol.26 No.3 March 2001
http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
151News&Comment
Journal Club
Inclusion bodies (IBs) are major proteinaggregates that arise owing to a deficiency ofthe cell’s protein folding machinery. This oftenoccurs in recombinant bacteria whereplasmid-encoded genes are being highlyexpressed. In this context, research into theprevention of IB formation or the re-formingof correctly folded protein from IBs is of greatimportance to the biotechnology industry.Understanding IBs is equally important forresearch into amyloid diseases (such asAlzheimer’s disease), which are caused bymisfolding of proteins, resulting in theirdeposition in the cells as fibrils. Surprisingly,given the significance of IBs, they havegenerally been poorly investigated. However,a recent paper by Carrió et al.1 is an exception.They have shown that IB production in vivo is,in fact, caused by an imbalance betweenprotein aggregation and solubility.
The authors used two plasmid-encodedproteins expressed in Escherichia coli to studyIB production. One was a β-galactosidasefused to a viral capsid protein, and the otherwas phage 22 tail-spike protein (TSP). In both
systems, they calculated the number of IBsformed and their volume, the amount ofsoluble versus insoluble protein, and theactivity of the protein as an indicator of properfolding. These measurements were taken athourly intervals before and after the additionof chloramphenicol, which arrests proteinsynthesis. In the β-galactosidase system,using SDS-PAGE and densitometry againstknown standards, they showed that theamount of insoluble material in the cellcultures sharply decreased upon addition ofchloramphenicol, with a concomitantincrease in the amount of soluble material.A combination of microscopy and digitalimaging also showed a reduction in thenumber and volume of IBs in the cells. Correctre-folding of the protein was confirmed by anincrease in the β-galactosidase activity in thecultures following termination of proteinsynthesis. The same trend was found with theexpression of TSP. Carrió et al. alsodemonstrated in vitro re-folding of IB proteinsby incubating purified IBs from the two strainswith cell extracts from plasmid-free BL26 cells,
with the result being a recovery of enzymaticactivity and soluble peptide in both systems.
The authors concluded that IBs are theresult of an imbalance in the equilibriumbetween protein precipitation and re-foldingand solubility in the cell. By switching offprotein production, the protein foldingmachinery can catch up, thus redressing thebalance. The authors therefore see IBs not asa dead-end product caused by inefficiency ofthe bacterial protein folding machinery, butrather as a reversible reservoir of proteinawaiting correct folding when conditionspermit. The significance of the result is alreadyapparent: IB solubility in vitro could providethe answer to the problem of IB formation inbiotechnology and, from a clinical viewpoint,it raises possibilities for new therapeuticapproaches to amyloid diseases.
1 Carrió, M.M. et al. (2000) Fine architecture ofbacterial inclusion bodies. FEBS Lett. 471, 7–11
James Pearce
Inclusion bodies as reversible protein reservoirs
Repair of DNA damage by the versatilenucleotide excision repair (NER) pathwayproceeds much faster in the transcribedstrand of an active gene than it does in thenon-transcribed strand or in the globalgenome. This transcription-coupled repair(TCR) was first described almost 15 years agobut, despite its obvious importance formaintenance of genome integrity, little hasbeen learned about the molecularmechanisms that make such preferentialrepair possible. New results from the Brouwerlaboratory1 now suggest that TCR worksbetter when the efficiency of transcriptionalelongation is relaxed.
Two genes, Cockayne’s Syndrome A(CSA) and B (CSB), encode proteins that areinvolved in this process. The yeasthomologue of CSB is RAD26 and, likemammalian CS cells, rad26 cells show amarkedly slower repair of the transcribedstrand of an active gene than do normal cells.CSB and Rad26 are DNA-dependent ATPases
often thought of as possible functionalanalogues of the bacterial transcriptioncoupling repair factor (TCRF) protein, whichinteracts directly with the bacterial NERmachinery and is capable of displacing astalled RNA polymerase, thus enabling DNArepair. The new results1 show that in anactive yeast gene, where TCR was previouslyshown to be completely dependent onRAD26, fast preferential repair of thetranscribed strand can actually take place inthe absence of RAD26, provided the SPT4gene is also deleted. Spt4 and its partnerSpt5 form a complex [DRB sensitivity-inducing factor (DSIF) in mammalian cells]that assists transcription elongation andinteracts with RNA polymerase II. Geneticinteractions between SPT4 and NER factorsindicate an even closer relationship betweentranscriptional elongation and NER thanpreviously described.
These data suggest that, in contrast toTCRF, Rad26 (and, thereby in all likelihood,
CSB) is not involved in the TCR reaction per se,and point to RNA polymerase II elongationcomplexes as important determinants for theefficiency of TCR. Different models to explainthe possible role of Rad26 in TCR could besuggested. However, one simpleinterpretation is that, in the presence of DNAdamage, efficient transcriptional elongationbecomes counter-productive. During suchadversity, Rad26 and CSB might help toprevent the RNA polymerase II elongationcomplexes from ‘choking’ on the damage.When Spt4 is missing from cells, transcriptionelongation efficiency might already bereduced, thus eliminating the need for Rad26and CSB.
1 Jansen, L.T. et al. (2000) Spt4 modulates Rad26requirement in transcription-coupled nucleotideexcision repair. EMBO J. 19, 6498–6507
Jesper Q. Svejstrup
Transcription-coupled DNA repair without the transcription-couplingrepair factor
