The Mammalian Disaggregase Machinery: Hsp110 Synergizes with Hsp70 and Hsp40 to Catalyze Protein Disaggregation and Reactivation in a Cell-Free System

Bacteria, fungi, protozoa, chromista and plants all harbor homologues of Hsp104, a AAA+ ATPase that collaborates with Hsp70 and Hsp40 to promote protein disaggregation and reactivation. Curiously, however, metazoa do not possess an Hsp104 homologue. Thus, whether animal cells renature large protein aggregates has long remained unclear. Here, it is established that mammalian cytosol prepared from different sources possesses a potent, ATP-dependent protein disaggregase and reactivation activity, which can be accelerated and stimulated by Hsp104. This activity did not require the AAA+ ATPase, p97. Rather, mammalian Hsp110 (Apg-2), Hsp70 (Hsc70 or Hsp70) and Hsp40 (Hdj1) were necessary and sufficient to slowly dissolve large disordered aggregates and recover natively folded protein. This slow disaggregase activity was conserved to yeast Hsp110 (Sse1), Hsp70 (Ssa1) and Hsp40 (Sis1 or Ydj1). Hsp110 must engage substrate, engage Hsp70, promote nucleotide exchange on Hsp70, and hydrolyze ATP to promote disaggregation of disordered aggregates. Similarly, Hsp70 must engage substrate and Hsp110, and hydrolyze ATP for protein disaggregation. Hsp40 must harbor a functional J domain to promote protein disaggregation, but the J domain alone is insufficient. Optimal disaggregase activity is achieved when the Hsp40 can stimulate the ATPase activity of Hsp110 and Hsp70. Finally, Hsp110, Hsp70 and Hsp40 fail to rapidly remodel amyloid forms of the yeast prion protein, Sup35, or the Parkinson's disease protein, alpha-synuclein. However, Hsp110, Hsp70 and Hsp40 enhanced the activity of Hsp104 against these amyloid substrates. Taken together, these findings suggest that Hsp110 fulfils a subset of Hsp104 activities in mammals. Moreover, they suggest that Hsp104 can collaborate with the mammalian disaggregase machinery to rapidly remodel amyloid conformers

Inactivation of VCP/ter94 Suppresses Retinal Pathology Caused by Misfolded Rhodopsin in Drosophila

The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (RhP23H). Unlike the wild-type Rh, mutant RhP23H exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded RhP23H and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that RhP23H is a substrate of the ER–associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1P37H (the equivalent of mammalian RhP23H) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1P37H toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1P37H and further activated the Ire1/Xbp1 ER stress pathway in the Rh1P37H retina. Despite this, Rh1P37H flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1P37H retina. Pharmacological treatment of Rh1P37H flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration