Background: Prefoldin, a molecular chaperone composed of six subunits, prevents misfolding of newly synthesized nascent polypeptides. Results: Prefoldin inhibited aggregation of pathogenic Huntingtin and subsequent cell death. Conclusion: Prefoldin suppressed Huntingtin aggregation at the small oligomer stage. Significance: Prefoldin plays a role in preventing protein aggregation in Huntington disease.

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/ CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1␣/PFD5 as a novel c-Myc-binding protein and found that MM-1␣ suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1␣ into cells increased the level of endogenous MM-1␣. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1␣ but not that of endogenous MM-1␣, indicating that overexpressed MM-1␣, but not endogenous MM-1␣, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after cotransfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. Molecular chaperones assist newly synthesized or denatured proteins to be naturally folded in cells (1). Prefoldin (PFD)2 was identified as an unfolded actin-binding protein (2) and was shown to play a role as a chaperone by recruiting substrates such as actins and tubulins to the eukaryotic cytosolic chaperonin TRiC/CCT, which facilitates the folding of proteins through ATP hydrolysis (3-6). It has been reported that deletion of genes encoding prefoldin subunits in yeast resulted in cytoskeletal defects and in a slow-growth phenotype (2, 7, 8), which are similar to those found in mutations in CCT subunits, suggesting that coordinated function of prefoldin and TRiC/ CCT is necessary for cells to maintain normal cytoskeletons.Elucidation of the crystal structure of Methanobacterium thermoautotrophicum prefoldin (9) and results of analysis of eukaryotic prefoldin by electron microscopy (10) revealed that prefoldin is a hexameric complex with a jellyfish-like structure and that each subunit forms a coiled-coil structure at the Nand C-terminal ␣ helices. Archaea prefoldin possesses only two subunits, prefoldin ␣ and ␤, and builds up a ␣2␤4 hexamer (11). Eukaryotic prefoldin, on the other hand, possess six subunits, two ␣ subunits (PFD3 and PFD5) and four ␤ subunits (PFD1, PFD2, PFD4, and PFD6) (9, 11). In both archaea and eukaryotes, prefoldin ␣ subunits contain two ␤ hairpins in connecting regions at both terminals, and the ␤ subunits contain one ␤ hairpin to a...

Protein aggregation is observed in various neurodegeneration diseases, including Parkinson's disease (PD). Alpha-synuclein, a causative gene product of familial PD, is a major component of large aggregates (inclusion bodies) in PD. Prefoldin, a molecular chaperone comprised of six subunits, PFD1~6, prevents misfolding of newly synthesized nascent polypeptides and also prevents aggregation of protein such as a pathogenic form of Huntingtin, a causative gene product of Huntington disease. In this study, we first found that aggregation of TagRFP-tagged wild-type -synuclein and its pathogenic mutants, but not that of GFP-tagged -synuclein,

We have reported that a novel c-Myc-binding protein, MM-1, repressed E-box-dependent transcription and transforming activities of c-Myc and that a mutation of A157R in MM-1, which is often observed in patients with leukemia or lymphoma, abrogated all of the repressive activities of MM-1 toward c-Myc, indicating that MM-1 is a novel tumor suppressor. MM-1 also binds to the ubiquitin-proteasome system, leading to degradation of c-Myc. In this study, we identified Rabring7, a Rab7-binding and RING finger-containing protein, as an MM-1-binding protein, and we found that Rabring7 mono-ubiquitinated MM-1 in the cytoplasm without degradation of MM-1. Rabring7 was also found to bind to c-Myc and to ubiquitinate c-Myc in a threonine 58-dependent manner. When c-Myc was co-transfected with MM-1 and Rabring7, c-Myc was degraded. Furthermore, it was found that c-Myc was stabilized in MM-1-knockdown cells even when Rabring7 was transfected and that Rabring7 was bound to and co-localized with MM-1 and c-Myc after MM-1 and Rabring7 had been translocated from the cytoplasm to the nucleus. These results suggest that Rabring7 stimulates c-Myc degradation via mono-ubiquitination of MM-1.

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