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Help for Protein Folding

Incorrectly folded proteins can wreak havoc inside a cell. Fortunately, all organisms possess chaperones, a group of proteins that assist with folding. Escherichia coli use the chaperone DnaK and its co-chaperones DnaJ and GrpE. Under conditions of heat stress, DnaK binds to misfolded proteins and undergoes conformational changes facilitated by DnaJ and GrpE. These changes cause the release of the now partially refolded protein which either spontaneously finishes folding or is rebound by chaperones. Under non-stress conditions, DnaJ binds to specific native proteins, such as P1 RepA, and facilitates their folding by targeting them to DnaK.

CpbM exists as a complex with CpbA.

E. coli also possesses a DnaJ homologue, CbpA, which can act as a multicopy suppressor for dnaJ mutations. Interested in seeing if CbpA behaves like a DnaJ-like co-chaperone, Chi Chae et al. looked at the function of CpbA. Using a series of in vitro experiments, the researchers showed that CbpA does possess some DnaJ co-chaperone abilities but differs from it in other respects. For example, CpbA can act with DnaK to prevent protein aggregation, and it can form a complex with RepA to target it to DnaK for remodeling, but it lacks the autonomous chaperone activity found in DnaJ.

Chae et al. also discovered that a protein they named CpbM (CbpA Modulator) is coexpressed with CpbA. CpbM is similar in amino acid sequence to DafA, a Thermus thermophilus chaperone that forms trimers with DnaK and DnaJ to inhibit their activities. The authors found that CpbM binds CpbA and blocks its co-chaperone activity, suggesting that, like DafA, it acts as a chaperone activity modulator.

FOOTNOTES

See referenced article, J. Biol. Chem. 2004, 279, 33147–33153

Sequestered Protein Reveals Its Targets

Cellular transcription is pre-dominantly controlled by the assembly of transcription factors into macromolecular complexes. The mammalian transcriptional coactivator HCF-1 is involved in the recruitment and assembly of these transcription complexes. HCF-1 is a nuclear protein that was originally identified for its role in interacting with the herpes protein VP16 during the assembly of the herpes simplex virus regulatory complex. Since then, HCF-1 has also been implicated in cell-cycle progression, chromatin remodeling, and mRNA processing.

Cells expressing the fusion protein (T-Act) show increases in levels of cytoplasmic HCF-1 over time as compared to control cells (MEP).

Because HCF-1 is required for cell viability, it has been difficult to identify its cellular transcriptional targets using conventional methods such as dominant-negative and siRNA knock-down approaches. Bharat Khurana and Thomas M. Kristie circumvented this problem by developing a genetic system to specifically sequester HCF-1 in the cytoplasm in a regulated manner. researchers fused actin to a high affinity HCF-1 interaction motif derived from VP16 added an inducible promoter. Cellular incorporation of the actin fusion protein into cytoplasmic actin filaments prevented the nuclear transport of HCF-1 by acting as a high anchor. The resulting cells had defects in herpes simplex virus gene expression and displayed reduced viral yields.

The researchers also used multiple microarray hybridizations with mRNA from HCF-1 sequestering cells to identify targets dependent on HCF-1 nuclear function. resulting 188 HCF-1-dependent genes encode a range of proteins involved in cell-cycle gression, apoptosis, development, DNA replication, RNA processing, transcription, and signal transduction.

FOOTNOTES

See referenced article, J. Biol. Chem. 2004, 279, 33673–33683