Mechanistic Insight into the Suppression of Polyglutamine Aggregation by SRCP1.

Protein aggregation is a hallmark of the polyglutamine diseases. One potential treatment for these diseases is suppression of polyglutamine aggregation. Previous work identified the cellular slime mold Dictyostelium discoideum as being naturally resistant to polyglutamine aggregation. Further work identified serine-rich chaperone protein 1 (SRCP1) as a protein that is both necessary in Dictyostelium and sufficient in human cells to suppress polyglutamine aggregation. Therefore, understanding how SRCP1 suppresses aggregation may be useful for developing therapeutics for the polyglutamine diseases. Here we utilized a de novo protein modeling approach to generate predictions of SRCP1's structure. Using our best-fit model, we generated mutants that were predicted to alter the stability of SRCP1 and tested these mutants' stability in cells. Using these data, we identified top models of SRCP1's structure that are consistent with the C-terminal region of SRCP1 forming a ß-hairpin with a highly dynamic N-terminal region. We next generated a series of peptides that mimic the predicted ß-hairpin and validated that they inhibit aggregation of a polyglutamine-expanded mutant huntingtin exon 1 fragment in vitro. To further assess mechanistic details of how SRCP1 inhibits polyglutamine aggregation, we utilized biochemical assays to determine that SRCP1 inhibits secondary nucleation in a manner dependent upon the regions flanking the polyglutamine tract. Finally, to determine if SRCP1 more could generally suppress protein aggregation, we confirmed that it was sufficient to inhibit aggregation of polyglutamine-expanded ataxin-3. Together these studies provide details into the structural and mechanistic basis of the inhibition of protein aggregation by SRCP1.

Viral vector gene delivery of the novel chaperone protein SRCP1 to modify insoluble protein in in vitro and in vivo models of ALS.

Protein misfolding and aggregation are shared features of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and protein quality control disruption contributes to neuronal toxicity. Therefore, reducing protein aggregation could hold therapeutic potential. We previously identified a novel chaperone protein, serine-rich chaperone protein 1 (SRCP1), that effectively prevents protein aggregation in cell culture and zebrafish models of Huntington's disease. Here we tested whether this benefit extends to aggregated proteins found in ALS. We used viral-mediated expression of SRCP1 in in vitro and in vivo models of ALS. We found that SRCP1 reduced insoluble SOD1 protein levels in HEK293T cells overexpressing either the A4V or G93R mutant SOD1. However, the reduction of insoluble protein was not observed in either mutant C9orf72 or SOD1 ALS iPSC-derived motor neurons infected with a lentivirus expressing SRCP1. SOD1-G93A ALS mice injected with AAV-SRCP1 showed a small but significant reduction in insoluble and soluble SOD1 in both the brain and spinal cord, but SRCP1 expression did not improve mouse survival. These data indicate that SRCP1 likely reduces insoluble protein burden in a protein and/or context-dependent manner indicating a need for additional insight into SRCP1 function and therapeutic potential.

Insights on Microsatellite Characteristics, Evolution, and Function From the Social Amoeba Dictyostelium discoideum.

Microsatellites are repetitive sequences commonly found in the genomes of higher organisms. These repetitive sequences are prone to expansion or contraction, and when microsatellite expansion occurs in the regulatory or coding regions of genes this can result in a number of diseases including many neurodegenerative diseases. Unlike in humans and other organisms, the social amoeba Dictyostelium discoideum contains an unusually high number of microsatellites. Intriguingly, many of these microsatellites fall within the coding region of genes, resulting in nearly 10,000 homopolymeric repeat proteins within the Dictyostelium proteome. Surprisingly, among the most common of these repeats are polyglutamine repeats, a type of repeat that causes a class of nine neurodegenerative diseases in humans. In this minireview, we summarize what is currently known about homopolymeric repeats and microsatellites in Dictyostelium discoideum and discuss the potential utility of Dictyostelium for identifying novel mechanisms that utilize and regulate regions of repetitive DNA.

Chemical Regulation of the Protein Quality Control E3 Ubiquitin Ligase C-Terminus of Hsc70 Interacting Protein (CHIP).

The ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) is an important regulator of proteostasis. Despite playing an important role in maintaining proteostasis, little progress has been made in developing small molecules that regulate ubiquitin transfer by CHIP. Here we used differential scanning fluorimetry to identify compounds that bound CHIP. Compounds that bound CHIP were then analyzed by quantitative ubiquitination assays to identify those that altered CHIP function. One compound, MS.001, inhibited both the chaperone binding and ubiquitin ligase activity of CHIP at low micromolar concentrations. Interestingly, we found that MS.001 did not have activity against isolated U-box or tetratricopeptide (TPR) domains, but instead only inhibited full-length CHIP. Using in silico docking we identified a potential MS.001 binding site on the linker domain of CHIP and mutation of this site rendered CHIP resistant to MS.001. Together our data identify an inhibitor of the E3 ligase CHIP and provides insight into the development of compounds that regulate CHIP activity.

Assessing the necessity of a family of genes that encode small proteins in Dictyostelium discoideum development.

Dictyostelium discoideum's genome encodes for a large class of small proteins that are developmentally regulated. We deleted six of the genes that encode these proteins to determine if they play an essential role in Dictyostelium discoideum development. Deletion of these genes had no significant effect on Dictyostelium discoideum development. These results suggest that the selected genes do not play an essential role in Dictyostelium discoideum development.

Dictyostelium discoideum as a Model for Investigating Neurodegenerative Diseases.

The social amoeba Dictyostelium discoideum is a model organism that is used to investigate many cellular processes including chemotaxis, cell motility, cell differentiation, and human disease pathogenesis. While many single-cellular model systems lack homologs of human disease genes, Dictyostelium's genome encodes for many genes that are implicated in human diseases including neurodegenerative diseases. Due to its short doubling time along with the powerful genetic tools that enable rapid genetic screening, and the ease of creating knockout cell lines, Dictyostelium is an attractive model organism for both interrogating the normal function of genes implicated in neurodegeneration and for determining pathogenic mechanisms that cause disease. Here we review the literature involving the use of Dictyostelium to interrogate genes implicated in neurodegeneration and highlight key questions that can be addressed using Dictyostelium as a model organism.

Development of a Positive Selection High Throughput Genetic Screen in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum is a powerful model organism that can be utilized to investigate human health and disease. One particular strength of Dictyostelium is that it can be utilized for high throughput genetic screens. For many phenotypes, one limitation of utilizing Dictyostelium is that screening can be an arduous and time-consuming process, limiting the genomic depth one can cover. Previously, we utilized a restriction enzyme-mediated integration screen to identify suppressors of polyglutamine aggregation in Dictyostelium. However, due to the time required to perform the screen, we only obtained ∼4% genome coverage. Here we have developed an efficient screening pipeline that couples chemical mutagenesis with the 5-fluoroorotic acid counterselection system to enrich for mutations in genes of interest. Here we describe this new screening methodology and highlight how it can be utilized for other biological systems.

A Heat Shock Protein 48 (HSP48) Biomolecular Condensate Is Induced during Dictyostelium discoideum Development.

The social amoeba Dictyostelium discoideum's proteome contains a vast array of simple sequence repeats, providing a unique model to investigate proteostasis. Upon conditions of cellular stress, D. discoideum undergoes a developmental process, transitioning from a unicellular amoeba to a multicellular fruiting body. Little is known about how proteostasis is maintained during D. discoideum's developmental process. Here, we have identified a novel α-crystallin domain-containing protein, heat shock protein 48 (HSP48), that is upregulated during D. discoideum development. HSP48 functions in part by forming a biomolecular condensate via its highly positively charged intrinsically disordered carboxy terminus. In addition to HSP48, the highly negatively charged primordial chaperone polyphosphate is also upregulated during D. discoideum development, and polyphosphate functions to stabilize HSP48. Upon germination, levels of both HSP48 and polyphosphate dramatically decrease, consistent with a role for HSP48 and polyphosphate during development. Together, our data demonstrate that HSP48 is strongly induced during Dictyostelium discoideum development. We also demonstrate that HSP48 forms a biomolecular condensate and that polyphosphate is necessary to stabilize the HSP48 biomolecular condensate.IMPORTANCE During cellular stress, many microbes undergo a transition to a dormant state. This includes the social amoeba Dictyostelium discoideum that transitions from a unicellular amoeba to a multicellular fruiting body upon starvation. In this work, we identify heat shock protein 48 (HSP48) as a chaperone that is induced during development. We also show that HSP48 forms a biomolecular condensate and is stabilized by polyphosphate. The findings here identify Dictyostelium discoideum as a novel microbe to investigate protein quality control pathways during the transition to dormancy.

SRCP1 Conveys Resistance to Polyglutamine Aggregation.

The polyglutamine (polyQ) diseases are a group of nine neurodegenerative diseases caused by the expansion of a polyQ tract that results in protein aggregation. Unlike other model organisms, Dictyostelium discoideum is a proteostatic outlier, naturally encoding long polyQ tracts yet resistant to polyQ aggregation. Here we identify serine-rich chaperone protein 1 (SRCP1) as a molecular chaperone that is necessary and sufficient to suppress polyQ aggregation. SRCP1 inhibits aggregation of polyQ-expanded proteins, allowing for their degradation via the proteasome, where SRCP1 is also degraded. SRCP1's C-terminal domain is essential for its activity in cells, and peptides that mimic this domain suppress polyQ aggregation in vitro. Together our results identify a novel type of molecular chaperone and reveal how nature has dealt with the problem of polyQ aggregation.

Interaction of the polyglutamine protein ataxin-3 with Rad23 regulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3.

Polyglutamine (polyQ) repeat expansion in the deubiquitinase ataxin-3 causes neurodegeneration in Spinocerebellar Ataxia Type 3 (SCA3), one of nine inherited, incurable diseases caused by similar mutations. Ataxin-3's degradation is inhibited by its binding to the proteasome shuttle Rad23 through ubiquitin-binding site 2 (UbS2). Disrupting this interaction decreases levels of ataxin-3. Since reducing levels of polyQ proteins can decrease their toxicity, we tested whether genetically modulating the ataxin-3-Rad23 interaction regulates its toxicity in Drosophila. We found that exogenous Rad23 increases the toxicity of pathogenic ataxin-3, coincident with increased levels of the disease protein. Conversely, reducing Rad23 levels alleviates toxicity in this SCA3 model. Unexpectedly, pathogenic ataxin-3 with a mutated Rad23-binding site at UbS2, despite being present at markedly lower levels, proved to be more pathogenic than a disease-causing counterpart with intact UbS2. Additional studies established that the increased toxicity upon mutating UbS2 stems from disrupting the autoprotective role that pathogenic ataxin-3 has against itself, which depends on the co-chaperone, DnaJ-1. Our data reveal a previously unrecognized balance between pathogenic and potentially therapeutic properties of the ataxin-3-Rad23 interaction; they highlight this interaction as critical for the toxicity of the SCA3 protein, and emphasize the importance of considering protein context when pursuing suppressive avenues.

Allosteric regulation of deubiquitylase activity through ubiquitination.

Ataxin-3, the protein responsible for spinocerebellar ataxia type-3, is a cysteine protease that specifically cleaves poly-ubiquitin chains and participates in the ubiquitin proteasome pathway. The enzymatic activity resides in the N-terminal Josephin domain. An unusual feature of ataxin-3 is its low enzymatic activity especially for mono-ubiquitinated substrates and short ubiquitin chains. However, specific ubiquitination at lysine 117 in the Josephin domain activates ataxin-3 through an unknown mechanism. Here, we investigate the effects of K117 ubiquitination on the structure and enzymatic activity of the protein. We show that covalently linked ubiquitin rests on the Josephin domain, forming a compact globular moiety and occupying a ubiquitin binding site previously thought to be essential for substrate recognition. In doing so, ubiquitination enhances enzymatic activity by locking the enzyme in an activated state. Our results indicate that ubiquitin functions both as a substrate and as an allosteric regulatory factor. We provide a novel example in which a conformational switch controls the activity of an enzyme that mediates deubiquitination.

A bipartite interaction between Hsp70 and CHIP regulates ubiquitination of chaperoned client proteins.

The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners.

Intrinsic disorder drives N-terminal ubiquitination by Ube2w.

Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells.

Ubiquitin-binding site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23.

Polyglutamine repeat expansion in ataxin-3 causes neurodegeneration in the most common dominant ataxia, spinocerebellar ataxia type 3 (SCA3). Since reducing levels of disease proteins improves pathology in animals, we investigated how ataxin-3 is degraded. Here we show that, unlike most proteins, ataxin-3 turnover does not require its ubiquitination, but is regulated by ubiquitin-binding site 2 (UbS2) on its N terminus. Mutating UbS2 decreases ataxin-3 protein levels in cultured mammalian cells and in Drosophila melanogaster by increasing its proteasomal turnover. Ataxin-3 interacts with the proteasome-associated proteins Rad23A/B through UbS2. Knockdown of Rad23 in cultured cells and in Drosophila results in lower levels of ataxin-3 protein. Importantly, reducing Rad23 suppresses ataxin-3-dependent degeneration in flies. We present a mechanism for ubiquitination-independent degradation that is impeded by protein interactions with proteasome-associated factors. We conclude that UbS2 is a potential target through which to enhance ataxin-3 degradation for SCA3 therapy.

Enzymatic production of mono-ubiquitinated proteins for structural studies: The example of the Josephin domain of ataxin-3.

Protein ubiquitination occurs through formation of an isopeptide bond between the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate lysine residue. This post-translational modification, which occurs through the attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin chains, is involved in crucial cellular events such as protein degradation, cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin pathways is also implicated in the pathogenesis of several human diseases ranging from cancer to neurodegeneration. However, despite the undoubted biological importance, understanding the molecular basis of how ubiquitination regulates different pathways has up to now been strongly limited by the difficulty of producing the amounts of highly homogeneous samples that are needed for a structural characterization by X-ray crystallography and/or NMR. Here, we report on the production of milligrams of highly pure Josephin mono-ubiquitinated on lysine 117 through large scale in vitro enzymatic ubiquitination. Josephin is the catalytic domain of ataxin-3, a protein responsible for spinocerebellar ataxia type 3. Ataxin-3 is the first deubiquitinating enzyme (DUB) reported to be activated by mono-ubiquitination. We demonstrate that the samples produced with the described method are correctly folded and suitable for structural studies. The protocol allows facile selective labelling of the components. Our results provide an important proof-of-concept that may pave the way to new approaches to the in vitro study of ubiquitinated proteins.

Ubiquitination regulates the neuroprotective function of the deubiquitinase ataxin-3 in vivo.

Deubiquitinases (DUBs) are proteases that regulate various cellular processes by controlling protein ubiquitination. Cell-based studies indicate that the regulation of the activity of DUBs is important for homeostasis and is achieved by multiple mechanisms, including through their own ubiquitination. However, the physiological significance of the ubiquitination of DUBs to their functions in vivo is unclear. Here, we report that ubiquitination of the DUB ataxin-3 at lysine residue 117, which markedly enhances its protease activity in vitro, is critical for its ability to suppress toxic protein-dependent degeneration in Drosophila melanogaster. Compared with ataxin-3 with only Lys-117 present, ataxin-3 that does not become ubiquitinated performs significantly less efficiently in suppressing or delaying the onset of toxic protein-dependent degeneration in flies. According to further studies, the C terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase that ubiquitinates ataxin-3 in vitro, is dispensable for its ubiquitination in vivo and is not required for the neuroprotective function of this DUB in Drosophila. Our work also suggests that ataxin-3 suppresses degeneration by regulating toxic protein aggregation rather than stability.

Accelerated neurodegeneration through chaperone-mediated oligomerization of tau.

Aggregation of tau protein in the brain is associated with a class of neurodegenerative diseases known as tauopathies. FK506 binding protein 51 kDa (FKBP51, encoded by FKBP5) forms a mature chaperone complex with Hsp90 that prevents tau degradation. In this study, we have shown that tau levels are reduced throughout the brains of Fkbp5-/- mice. Recombinant FKBP51 and Hsp90 synergized to block tau clearance through the proteasome, resulting in tau oligomerization. Overexpression of FKBP51 in a tau transgenic mouse model revealed that FKBP51 preserved the species of tau that have been linked to Alzheimer's disease (AD) pathogenesis, blocked amyloid formation, and decreased tangle load in the brain. Alterations in tau turnover and aggregate structure corresponded with enhanced neurotoxicity in mice. In human brains, FKBP51 levels increased relative to age and AD, corresponding with demethylation of the regulatory regions in the FKBP5 gene. We also found that higher FKBP51 levels were associated with AD progression. Our data support a model in which age-associated increases in FKBP51 levels and its interaction with Hsp90 promote neurotoxic tau accumulation. Strategies aimed at attenuating FKBP51 levels or its interaction with Hsp90 have the potential to be therapeutically relevant for AD and other tauopathies.

The E3 ubiquitin ligase CHIP and the molecular chaperone Hsc70 form a dynamic, tethered complex.

The E3 ubiquitin ligase CHIP (C-terminus of Hsc70 Interacting Protein, a 70 kDa homodimer) binds to the molecular chaperone Hsc70 (a 70 kDa monomer), and this complex is important in both the ubiquitination of Hsc70 and the turnover of Hsc70-bound clients. Here we used NMR spectroscopy, biolayer interferometry, and fluorescence polarization to characterize the Hsc70-CHIP interaction. We found that CHIP binds tightly to two molecules of Hsc70 forming a 210 kDa complex, with a Kd of approximately 60 nM, and that the IEEVD motif at the C-terminus of Hsc70 (residues 642-646) is both necessary and sufficient for binding. Moreover, the same motif is required for CHIP-mediated ubiquitination of Hsc70 in vitro, highlighting its functional importance. Relaxation-based NMR experiments on the Hsc70-CHIP complex determined that the two partners move independently in solution, similar to "beads on a string". These results suggest that a dynamic C-terminal region of Hsc70 provides for flexibility between CHIP and the chaperone, allowing the ligase to "search" a large space and engage in productive interactions with a wide range of clients. In support of this suggestion, we find that deleting residues 623-641 of the C-terminal region, while retaining the IEEVD motif, caused a significant decrease in the efficiency of Hsc70 ubiquitination by CHIP.

CGG repeat-associated translation mediates neurodegeneration in fragile X tremor ataxia syndrome.

Fragile X-associated tremor ataxia syndrome (FXTAS) results from a CGG repeat expansion in the 5' UTR of FMR1. This repeat is thought to elicit toxicity as RNA, yet disease brains contain ubiquitin-positive neuronal inclusions, a pathologic hallmark of protein-mediated neurodegeneration. We explain this paradox by demonstrating that CGG repeats trigger repeat-associated non-AUG-initiated (RAN) translation of a cryptic polyglycine-containing protein, FMRpolyG. FMRpolyG accumulates in ubiquitin-positive inclusions in Drosophila, cell culture, mouse disease models, and FXTAS patient brains. CGG RAN translation occurs in at least two of three possible reading frames at repeat sizes ranging from normal (25) to pathogenic (90), but inclusion formation only occurs with expanded repeats. In Drosophila, CGG repeat toxicity is suppressed by eliminating RAN translation and enhanced by increased polyglycine protein production. These studies expand the growing list of nucleotide repeat disorders in which RAN translation occurs and provide evidence that RAN translation contributes to neurodegeneration.