Selective macroautophagy (autophagy) of ubiquitinated protein is implicated as a compensatory mechanism of the ubiquitin-proteasome system. p62/SQSTM1 is a key molecule managing autophagic clearance of polyubiquitinated proteins. However, little is known about mechanisms controlling autophagic degradation of polyubiquitinated proteins. Here, we show that the specific phosphorylation of p62 at serine 403 (S403) in its ubiquitin-associated (UBA) domain increases the affinity between UBA and polyubiquitin chain, resulting in efficiently targeting polyubiquitinated proteins in "sequestosomes" and stabilizing sequestosome structure as a cargo of ubiquitinated proteins for autophagosome entry. Casein kinase 2 (CK2) phosphorylates S403 of p62 directly. Furthermore, CK2 overexpression or phosphatase inhibition reduces the formation of inclusion bodies of the polyglutamine-expanded huntingtin exon1 fragment in a p62-dependent manner. We propose that phosphorylation of p62 at S403 regulates autophagic clearance of ubiquitinated proteins and protein aggregates that are poorly degraded by proteasomes.

Alterations in protein folding and the regulation of conformational states have become increasingly important to the functionality of key molecules in signaling, cell growth, and cell death. Molecular chaperones, because of their properties in protein quality control, afford conformational flexibility to proteins and serve to integrate stress-signaling events that influence aging and a range of diseases including cancer, cystic fibrosis, amyloidoses, and neurodegenerative diseases. We describe here characteristics of celastrol, a quinone methide triterpene and an active component from Chinese herbal medicine identified in a screen of bioactive small molecules that activates the human heat shock response. From a structure/function examination, the celastrol structure is remarkably specific and activates heat shock transcription factor 1 (HSF1) with kinetics similar to those of heat stress, as determined by the induction of HSF1 DNA binding, hyperphosphorylation of HSF1, and expression of chaperone genes. Celastrol can activate heat shock gene transcription synergistically with other stresses and exhibits cytoprotection against subsequent exposures to other forms of lethal cell stress. These results suggest that celastrols exhibit promise as a new class of pharmacologically active regulators of the heat shock response.

Proteins with expanded polyQ repeats are associated with at least nine neurodegenerative disorders including ataxins 1 and 3, Kennedy's disease, and Huntington's disease (HD) 1,2 . These diseases are dominantly inherited and although the polyQ-containing proteins are expressed widely in the brain, they result in selective neuronal death. There is a significant and striking correlation between the length of the polyQ repeat and pathology; longer repeats result in earlier onset and more severe symptoms with the threshold of approximately 40 glutamine residues. In the case of HD, for example, expanded polyQ in huntingtin (htt) protein causes disease 3,4 . A characteristic of the polyQ diseases is the appearance of neuronal inclusions that are formed by aggregation of the polyQ proteins with other cellular proteins 5,6 . This has led to the "toxic gain-of-function" hypothesis that essential proteins can be sequestered, which 3 over time leads to cellular dysgenesis. The expression of polyQ can cause other metastable proteins to lose functionality, and in turn these proteins amplify the toxicity of polyQ by enhancing overall aggregation 7 . Self-aggregation of polyQ proteins has been proposed to be mediated by association of parallel β-sheet structures 8 . However, the intrinsic in vivo events leading to the aggregation of polyQ proteins remain poorly understood.Protein misfolding is a natural consequence of protein biogenesis. To combat cytotoxicity that results from the accumulation of misfolded proteins, all cells express molecular chaperones that are essential for the productive folding of proteins 9,10 . Molecular chaperones are of several classes; for example, Hsp70/J-domain proteins interact with unfolded or partially folded proteins in concert with co-chaperones, while the chaperone machines of the chaperonin (Hsp60) family form cage-like structures that sequester non-native states of proteins 11 . The chaperonin containing t-complex polypeptide 1 (CCT)/t-complex polypeptide 1 ring complex (TRiC) is a member of chaperonin family 12 that facilitates the folding of proteins in the eukaryotic cytosol upon ATP hydrolysis 13,14 . CCT shows a weak but significant homology to E. coli GroEL and forms a hexadecamer double-troidal complex composed of eight different subunits 15,16 . Substrate proteins are captured in the cavity, and released after folding is completed 17 . Approximately 10% of newly-synthesized proteins have been proposed to be recognized by CCT.Recently, in a genome-wide screen to identify modifier genes for polyQ aggregation in C. elegans, approximately 200 genes were found to be required for the prevention of polyQ aggregation 18 . This included the genes encoding two Hsp70s, one J-protein, and six CCT subunits. These observations suggested a 4 role of CCT in preventing polyQ aggregation. We show here in mammalian cells that CCT has a key protective role against the toxicity of htt/polyQ and affects aggregation process at the soluble stage. In the context of our recent in vitro data showing that ...

Selective autophagy adaptor proteins, including p62/SQSTM1, play pivotal roles in the targeted degradation of ubiquitinated proteins or organelles through the autophagy-lysosome system. However, how autophagy adaptors promote the autophagosomal engulfment of selected substrates is poorly understood. Here, we show that p62 phosphorylation at S403 is required for the efficient autophagosomal engulfment of polyubiquitinated mitochondria during Parkin-dependent mitophagy. p62 is able to interact with Parkin-recruited mitochondria without S403 phosphorylation under mitophagy-inducing conditions, but those mitochondria are not enclosed by autophagosomes. Intriguingly, the S403 phosphorylation occurs only in the early period of mitochondrial depolarization. Optineurin and TANK-binding kinase 1 (TBK1) are transiently recruited to the polyubiquitinated mitochondria, and the activated TBK1 phosphorylates p62 at S403. TBK1 inhibitor, BX795, prevents the p62-mediated autophagosomal engulfment of Parkin-recruited mitochondria. Our results suggest that TBK1-mediated S403 phosphorylation regulates the efficient autophagosomal engulfment of ubiquitinated mitochondria as an immediate response to the mitochondrial depolarization.

Huntington's Disease (HD) is a dominantly inherited pathology caused by the accumulation of mutant huntingtin protein (HTT) containing an expanded polyglutamine (polyQ) tract. As the polyglutamine binding peptide 1 (QBP1) is known to bind an expanded polyQ tract but not the polyQ motif found in normal HTT, we selectively targeted mutant HTT for degradation by expressing a fusion molecule comprising two copies of QBP1 and copies of two different heat shock cognate protein 70 (HSC70)-binding motifs in cellular and mouse models of HD. Chaperone-mediated autophagy contributed to the specific degradation of mutant HTT in cultured cells expressing the construct. Intrastriatal delivery of a virus expressing the fusion molecule ameliorated the disease phenotype in the R6/2 mouse model of HD. Similar adaptor molecules comprising HSC70-binding motifs fused to an appropriate structure-specific binding agent(s) may have therapeutic potential for treating diseases caused by misfolded proteins other than those with expanded polyQ tracts.

SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxyterminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion, while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex. We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo.

The appearance of protein aggregates is a characteristic of protein misfolding disorders including familial amyotrophic lateral sclerosis, a neurodegenerative disease caused by inherited mutations in Cu/Zn superoxide dismutase 1 (SOD1). Here, we use live cell imaging of neuronal and nonneuronal cells to show that SOD1 mutants (G85R and G93A) form an aggregate structure consisting of immobile scaffolds, through which noninteracting cellular proteins can diffuse. Hsp70 transiently interacts, in a chaperone activity-dependent manner, with these mutant SOD1 aggregate structures. In contrast, the proteasome is sequestered within the aggregate structure, an event associated with decreased degradation of a proteasomal substrate. Through the use of time-lapse microscopy of individual cells, we show that nearly all (90%) aggregate-containing cells express higher levels of mutant SOD1 and died within 48 h, whereas 70% of cells expressing a soluble mutant SOD1 survived. Our results demonstrate that SOD1 G85R and G93A mutants form a distinct class of aggregate structures in cells destined for neuronal cell death.

The entorhinal cortex provides the major cortical input to the hippocampus, and both structures have been implicated in memory processes. The dynamics of neuronal circuits in the entorhinal-hippocampal system were studied in slices by optical imaging with high spatial and temporal resolution. Reverberation of neural activity was detected in the entorhinal cortex and was more prominent when the inhibition due to gamma-aminobutyric acid was slightly suppressed. Neural activity was transferred in a frequency-dependent way from the entorhinal cortex to the hippocampus. The entorhinal neuronal circuit could contribute to memory processes by holding information and selectively gating the entry of information into the hippocampus.