Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (HTT). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using in vitro and in vivo assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J-protein) that completely suppresses fibrilization of HttExon1Q48 The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient-derived neural cells and on an organismal level in Caenorhabditis elegans Among the proteins in this chaperone complex, the J-protein is the concentration-limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q97 aggregation and represents a target of future therapeutic avenues for HD.

Global Proteome and Ubiquitinome Changes in the Soluble and Insoluble Fractions of Q175 Huntington Mice Brains.

Huntington's disease is caused by a polyglutamine repeat expansion in the huntingtin protein which affects the function and folding of the protein, and results in intracellular protein aggregates. Here, we examined whether this mutation leads to altered ubiquitination of huntingtin and other proteins in both soluble and insoluble fractions of brain lysates of the Q175 knock-in Huntington's disease mouse model and the Q20 wild-type mouse model. Ubiquitination sites are detected by identification of Gly-Gly (diGly) remnant motifs that remain on modified lysine residues after digestion. We identified K6, K9, K132, K804, and K837 as endogenous ubiquitination sites of soluble huntingtin, with wild-type huntingtin being mainly ubiquitinated at K132, K804, and K837. Mutant huntingtin protein levels were strongly reduced in the soluble fraction whereas K6 and K9 were mainly ubiquitinated. In the insoluble fraction increased levels of huntingtin K6 and K9 diGly sites were observed for mutant huntingtin as compared with wild type. Besides huntingtin, proteins with various roles, including membrane organization, transport, mRNA processing, gene transcription, translation, catabolic processes and oxidative phosphorylation, were differently expressed or ubiquitinated in wild-type and mutant huntingtin brain tissues. Correlating protein and diGly site fold changes in the soluble fraction revealed that diGly site abundances of most of the proteins were not related to protein fold changes, indicating that these proteins were differentially ubiquitinated in the Q175 mice. In contrast, both the fold change of the protein level and diGly site level were increased for several proteins in the insoluble fraction, including ubiquitin, ubiquilin-2, sequestosome-1/p62 and myo5a. Our data sheds light on putative novel proteins involved in different cellular processes as well as their ubiquitination status in Huntington's disease, which forms the basis for further mechanistic studies to understand the role of differential ubiquitination of huntingtin and ubiquitin-regulated processes in Huntington's disease.

Prey for the Proteasome: Targeted Protein Degradation-A Medicinal Chemist's Perspective.

Targeted protein degradation (TPD), the ability to control a proteins fate by triggering its degradation in a highly selective and effective manner, has created tremendous excitement in chemical biology and drug discovery within the past decades. The TPD field is spearheaded by small molecule induced protein degradation with molecular glues and proteolysis targeting chimeras (PROTACs) paving the way to expand the druggable space and to create a new paradigm in drug discovery. However, besides the therapeutic angle of TPD a plethora of novel techniques to modulate and control protein levels have been developed. This enables chemical biologists to better understand protein function and to discover and verify new therapeutic targets. This Review gives a comprehensive overview of chemical biology techniques inducing TPD. It explains the strengths and weaknesses of these methods in the context of drug discovery and discusses their future potential from a medicinal chemist's perspective.

Expanded polyglutamine-containing N-terminal huntingtin fragments are entirely degraded by mammalian proteasomes.

Huntington disease is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat within the protein huntingtin (Htt). N-terminal fragments of the mutant Htt (mHtt) proteins containing the polyQ repeat are aggregation-prone and form intracellular inclusion bodies. Improving the clearance of mHtt fragments by intracellular degradation pathways is relevant to obviate toxic mHtt species and subsequent neurodegeneration. Because the proteasomal degradation pathway has been the subject of controversy regarding the processing of expanded polyQ repeats, we examined whether the proteasome can efficiently degrade Htt-exon1 with an expanded polyQ stretch both in neuronal cells and in vitro. Upon targeting mHtt-exon1 to the proteasome, rapid and complete clearance of mHtt-exon1 was observed. Proteasomal degradation of mHtt-exon1 was devoid of polyQ peptides as partial cleavage products by incomplete proteolysis, indicating that mammalian proteasomes are capable of efficiently degrading expanded polyQ sequences without an inhibitory effect on the proteasomal activity.

The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides.

Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington's disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein aggregation, preventing polyQ peptide aggregation by chaperones should greatly improve polyQ clearance and prevent aggregate formation. Here we expressed polyQ peptides in cells and show that their intracellular aggregation is prevented by DNAJB6 and DNAJB8, members of the DNAJ (Hsp40) chaperone family. In contrast, HSPA/Hsp70 and DNAJB1, also members of the DNAJ chaperone family, did not prevent peptide-initiated aggregation. Intriguingly, DNAJB6 and DNAJB8 also affected the soluble levels of polyQ peptides, indicating that DNAJB6 and DNAJB8 inhibit polyQ peptide aggregation directly. Together with recent data showing that purified DNAJB6 can suppress fibrillation of polyQ peptides far more efficiently than polyQ expanded protein fragments in vitro, we conclude that the mechanism of DNAJB6 and DNAJB8 is suppression of polyQ protein aggregation by directly binding the polyQ tract.

Identification of Small Molecules that Modulate Mutant p53 Condensation.

Structural mutants of p53 induce global p53 protein destabilization and misfolding, followed by p53 protein aggregation. First evidence indicates that p53 can be part of protein condensates and that p53 aggregation potentially transitions through a condensate-like state. We show condensate-like states of fluorescently labeled structural mutant p53 in the nucleus of living cancer cells. We furthermore identified small molecule compounds that interact with the p53 protein and lead to dissolution of p53 structural mutant condensates. The same compounds lead to condensation of a fluorescently tagged p53 DNA-binding mutant, indicating that the identified compounds differentially alter p53 condensation behavior depending on the type of p53 mutation. In contrast to p53 aggregation inhibitors, these compounds are active on p53 condensates and do not lead to mutant p53 reactivation. Taken together our study provides evidence for structural mutant p53 condensation in living cells and tools to modulate this process.

Crosstalk Between Chaperone-Mediated Protein Disaggregation and Proteolytic Pathways in Aging and Disease.

A functional protein quality control machinery is crucial to maintain cellular and organismal physiology. Perturbation in the protein homeostasis network can lead to the formation of misfolded and aggregated proteins that are a hallmark of protein conformational disorders and aging. Protein aggregation is counteracted by the action of chaperones that can resolubilize aggregated proteins. An alternative protein aggregation clearance strategy is the elimination by proteolysis employing the ubiquitin proteasome system (UPS) or autophagy. Little is known how these three protein aggregate clearance strategies are regulated and coordinated in an organism with the progression of aging or upon expression of disease-associated proteins. To unravel the crosstalk between the protein aggregate clearance options, we investigated how autophagy and the UPS respond to perturbations in protein disaggregation capacity. We found that autophagy is induced as a potential compensatory mechanism, whereas the UPS exhibits reduced capacity upon depletion of disaggregating chaperones in C. elegans and HEK293 cells. The expression of amyloid proteins Aß3-42 and Q40 result in an impairment of autophagy as well as the UPS within the same and even across tissues. Our data indicate a tight coordination between the different nodes of the proteostasis network (PN) with the progression of aging and upon imbalances of the capacity of each clearance mechanism.

Dynamic recruitment of ubiquitin to mutant huntingtin inclusion bodies.

Many neurodegenerative diseases, such as Huntington's disease, are hallmarked by the formation of intracellular inclusion bodies (IBs) that are decorated with ubiquitin, proteasomes and chaperones. The apparent enrichment of ubiquitin and components involved in protein quality control at IBs suggests local ubiquitin-dependent enzymatic activity. In this study, we examine recruitment of ubiquitin to IBs of polyglutamine-expanded huntingtin fragments (mHtt) by using synthesized TAMRA-labeled ubiquitin moieties. We show that intracellular TAMRA-ubiquitin is dynamic at mHtt IBs and is incorporated into poly-ubiquitin chains of intracellular substrates, such as mHtt, in a conjugation-dependent manner. Furthermore, we report that mHtt IBs recruit catalytically active enzymes involved in (de)-ubiquitination processes based on novel activity-based probes. However, we also find that the overexpression of the GFP-ubiquitin reporter, unlike the endogenous ubiquitin and TAMRA-ubiquitin, becomes irreversibly sequestered as a ring-like structure around the mHtt IBs, suggesting a methodical disadvantage of GFP-tagged ubiquitin. Our data provide supportive evidence for dynamic recruitment of ubiquitin and ubiquitin (de)-conjugating activity at mHtt initiated IBs.

Characterization of huntingtin pathologic fragments in human Huntington disease, transgenic mice, and cell models.

Huntington disease (HD) is caused by the expansion of a glutamine (Q) repeat near the N terminus of huntingtin (htt), resulting in altered conformation of the mutant protein to produce, most prominently in brain neurons, nuclear and cytoplasmic inclusion pathology. The inclusions and associated diffuse accumulation of mutant htt in nuclei are composed of N-terminal fragments of mutant protein. Here, we used a panel of peptide antibodies to characterize the htt protein pathologies in brain tissues from human HD, and a transgenic mouse model created by expressing the first 171 amino acids of human htt with 82Q (htt-N171-82Q). In tissues from both sources, htt pathologic features in nuclei were detected by antibodies to htt peptides 1-17 and 81-90 but not 115-129 (wild-type huntingtin numbering with 23 repeats). Human HEK 293 cells transfected with expression vectors that encode either the N-terminal 233 amino acids of human htt (htt-N233-82Q) or htt-N171-18Q accumulated smaller N-terminal fragments with antibody-binding characteristics identical to those of pathologic peptides. We conclude that the mutant htt peptides that accumulate in pathologic structures of human HD and httN171-82Q in mice are produced by similar, yet to be defined, proteolytic events in a region of the protein near or within amino acids 90-115.

Cellular strategies to cope with protein aggregation.

Nature has evolved several mechanisms to detoxify intracellular protein aggregates that arise upon proteotoxic challenges. These include the controlled deposition of misfolded proteins at distinct cellular sites, the protein disaggregation and refolding by molecular chaperones and/or degradation of misfolded and aggregated protein species by cellular clearance pathways. In this article, we discuss cellular the strategies of prokaroytes and eukaryotes to control protein aggregation.

Detection of ubiquitinated huntingtin species in intracellular aggregates.

Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins. Huntington's disease (HD) is one of nine diseases caused by an expanded polyQ repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin (Htt) fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated Htt such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated Htt fragments offers an important possibility to understand Htt degradation and aggregation processes within the cell. For the identification of aggregated Htt and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies.

Neurodegenerative disorders such as Huntington's disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that the proteasomes may become clogged by the aggregated protein fragments, leading to impairment of the ubiquitin-proteasome system. Here, we show by fluorescence pulse-chase experiments in living cells that proteasomes are dynamically and reversibly recruited into inclusion bodies. As these recruited proteasomes remain catalytically active and accessible to substrates, our results challenge the concept of proteasome sequestration and impairment in Huntington's disease, and support the reported absence of proteasome impairment in mouse models of Huntington's disease.

Alternative macroautophagic pathways.

Macroautophagy is a bulk degradation process that mediates the clearance of long-lived proteins, aggregates, or even whole organelles. This process includes the formation of autophagosomes, double-membrane structures responsible for delivering cargo to lysosomes for degradation. Currently, other alternative autophagy pathways have been described, which are independent of macroautophagic key players like Atg5 and Beclin 1 or the lipidation of LC3. In this review, we highlight recent insights in indentifying and understanding the molecular mechanism responsible for alternative autophagic pathways.

The Ubiquitin-Proteasome System in Huntington's Disease: Are Proteasomes Impaired, Initiators of Disease, or Coming to the Rescue?

Huntington's disease is a progressive neurodegenerative disease, caused by a polyglutamine expansion in the huntingtin protein. A prominent hallmark of the disease is the presence of intracellular aggregates initiated by N-terminal huntingtin fragments containing the polyglutamine repeat, which recruit components of the ubiquitin-proteasome system. While it is commonly thought that proteasomes are irreversibly sequestered into these aggregates leading to impairment of the ubiquitin-proteasome system, the data on proteasomal impairment in Huntington's disease is contradictory. In addition, it has been suggested that proteasomes are unable to actually cleave polyglutamine sequences in vitro, thereby releasing aggregation-prone polyglutamine peptides in cells. Here, we discuss how the proteasome is involved in the various stages of polyglutamine aggregation in Huntington's disease, and how alterations in activity may improve clearance of mutant huntingtin fragments.

Modulation of mutant huntingtin N-terminal cleavage and its effect on aggregation and cell death.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion near the N-terminus of huntingtin. A neuropathological hallmark of Huntington's disease is the presence of intracellular aggregates composed of mutant huntingtin N-terminal fragments in human postmortem brain, animal models, and cell culture models. It has been found that N-terminal fragments of the mutant huntingtin protein are more toxic than the full-length protein. Therefore, proteolytic processing of mutant huntingtin may play a key event in the pathogenesis of HD. Here, we present evidence that the region in huntingtin covering amino acids 116 to 125 is critical for N-terminal proteolytic processing. Within this region, we have identified mutations that either strongly reduce or enhance N-terminal cleavage. We took advantage of this effect and demonstrate that the mutation Δ121-122 within the putative cleavage region enhances N-terminal cleavage of huntingtin and the aggregation of N-terminal fragments. Furthermore, this particular deletion increased the activation of apoptotic processes and decreased neuronal cell viability. Our data indicate that the N-terminal proteolytic processing of mutant huntingtin can be modulated with an effect on aggregation and cell death rate.