Btn3 is a negative regulator of Btn2-mediated endosomal protein trafficking and prion curing in yeast

Yeast Btn2 facilitates the retrieval of specific proteins from late endosomes (LEs) to the Golgi, a process that may be adversely affected in Batten disease patients. We isolated the putative yeast orthologue of a human complex I deficiency gene, designated here as BTN3, as encoding a Btn2-interacting protein and negative regulator. First, yeast overexpressing BTN3 phenocopy the deletion of BTN2 and mislocalize certain trans-Golgi proteins, like Kex2 and Yif1, to the LE and vacuole, respectively. In contrast, the deletion of BTN3 results in a tighter pattern of protein localization to the Golgi. Second, BTN3 overexpression alters Btn2 localization from the IPOD compartment, which correlates with a sharp reduction in Btn2-mediated [URE3] prion curing. Third, Btn3 and the Snc1 v-SNARE compete for the same binding domain on Btn2, and this competition controls Btn2 localization and function. The inhibitory effects upon protein retrieval and prion curing suggest that Btn3 sequesters Btn2 away from its substrates, thus down-regulating protein trafficking and aggregation. Therefore Btn3 is a novel negative regulator of intracellular protein sorting, which may be of importance in the onset of complex I deficiency and Batten disease in humans.     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

Expression of human FUS/TLS in yeast leads to protein aggregation and cytotoxicity,recapitulating key features of FUS proteinopathy

Mutations in the fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene have been associated with amyotrophic lateral sclerosis (ALS). FUS-positive neuropathology is reported in a range of neurodegenerative diseases, including ALS and fronto-temporal lobar degeneration with ubiquitin-positive pathology (FTLD-U). To examine protein aggregation and cytotoxicity, we expressed human FUS protein in yeast. Expression of either wild type or ALS-associated R524S or P525L mutant FUS in yeast cells led to formation of aggregates and cytotoxicity, with the two ALS mutants showing increased cytotoxicity. Therefore, yeast cells expressing human FUS protein recapitulate key features of FUS-positive neurodegenerative diseases. Interestingly, a significant fraction of FUS expressing yeast cells stained by propidium iodide were without detectable protein aggregates, suggesting that membrane impairment and cellular damage caused by FUS expression may occur before protein aggregates become microscopically detectable and that aggregate formation might protect cells from FUS-mediated cytotoxicity. The N-terminus of FUS, containing the QGSY and G rich regions, is sufficient for the formation of aggregates but not cytotoxicity. The C-terminal domain, which contains a cluster of mutations, did not show aggregation or cytotoxicity. Similar to TDP-43 when expressed in yeast, FUS protein has the intrinsic property of forming aggregates in the absence of other human proteins. On the other hand, the aggregates formed by FUS are thioflavin T-positive and resistant to 0.5% sarkosyl, unlike TDP-43 when expressed in yeast cells. Furthermore, TDP-43 and FUS display distinct domain requirements in aggregate formation and cytotoxicity.     

Unexpected link between anaphase promoting complex and the toxicity of expanded polyglutamines expressed in yeast

Protein aggregation is intimately linked to a number of neurodegenerative diseases. Expansion of the huntingtin polyglutamine-rich domain causes protein aggregation and neuronal degeneration. Recently we found that, similar to neurons, yeast expressing the expanded domain show markers of programmed cell death. Here we showed that deletion of yeast metacaspase gene YCA1 partly rescues the toxic effect of the domain overexpression. We also performed genetic screen for other genes deletions alleviating the toxic effect and found ASE1. Ase1 is a substrate of the Cdh1 form of anaphase promoting complex, APC/Cdh1. We tested Cdh1 overexpression and the deletion of CLB2 (mitotic cyclin, substrate of APC/Cdh1) and found that both mutations had a rescuing effect on the expanded polyglutamine toxicity. Our data suggest that the toxic effect of aggregated proteins is partly indirect. We speculate that cellular attempt to degrade the aggregates overloads the proteasome, and this leads to pathological accumulation of APC substrates.     

Potentiated Hsp104 variants suppress toxicity of diverse neurodegenerative disease-linked proteins

Protein misfolding is implicated in numerous lethal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD). There are no therapies that reverse these protein-misfolding events. We aim to apply Hsp104, a hexameric AAA+ protein from yeast, to target misfolded conformers for reactivation. Hsp104 solubilizes disordered aggregates and amyloid, but has limited activity against human neurodegenerative disease proteins. Thus, we have previously engineered potentiated Hsp104 variants that suppress aggregation, proteotoxicity and restore proper protein localization of ALS and PD proteins in Saccharomyces cerevisiae, and mitigate neurodegeneration in an animal PD model. Here, we establish that potentiated Hsp104 variants possess broad substrate specificity and, in yeast, suppress toxicity and aggregation induced by wild-type TDP-43, FUS and α-synuclein, as well as missense mutant versions of these proteins that cause neurodegenerative disease. Potentiated Hsp104 variants also rescue toxicity and aggregation of TAF15 but not EWSR1, two RNA-binding proteins with a prion-like domain that are connected with the development of ALS and frontotemporal dementia. Thus, potentiated Hsp104 variants are not entirely non-specific. Indeed, they do not unfold just any natively folded protein. Rather, potentiated Hsp104 variants are finely tuned to unfold proteins bearing short unstructured tracts that are not recognized by wild-type Hsp104. Our studies establish the broad utility of potentiated Hsp104 variants.KEY WORDS: FUS, Hsp104, TDP-43, α-synuclein, Disaggregase, Neurodegeneration     

Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS

TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.     

Structural insights into the ubiquitin recognition by OPTN (optineurin) and its regulation by TBK1-mediated phosphorylation

OPTN (optineurin), a ubiquitin-binding scaffold protein, functions as an important macroautophagy/autophagy receptor in selective autophagy processes. Mutations in OPTN have been linked with human neurodegenerative diseases including ALS and glaucoma. However, the mechanistic basis underlying the recognition of ubiquitin by OPTN and its regulation by TBK1-mediated phosphorylation are still elusive. Here, we demonstrate that the UBAN domain of OPTN preferentially recognizes linear ubiquitin chain and forms an asymmetric 2:1 stoichiometry complex with the linear diubiquitin. In addition, our results provide new mechanistic insights into how phosphorylation of UBAN would regulate the ubiquitin-binding ability of OPTN and how disease-associated mutations in the OPTN UBAN domain disrupt its interaction with ubiquitin. Finally, we show that defects in ubiquitin-binding may affect the recruitment of OPTN to linear ubiquitin-decorated mutant Huntington protein aggregates. Taken together, our findings clarify the interaction mode between UBAN and linear ubiquitin chain in general, and expand our knowledge of the molecular mechanism of ubiquitin-decorated substrates recognition by OPTN as well as the pathogenesis of neurodegenerative diseases caused by OPTN mutations.     

Proteostatic imbalance and protein spreading in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder whose exact causative mechanisms are still under intense investigation. Several lines of evidence suggest that the anatomical and temporal propagation of pathological protein species along the neural axis could be among the main driving mechanisms for the fast and irreversible progression of ALS pathology. Many ALS‐associated proteins form intracellular aggregates as a result of their intrinsic prion‐like properties and/or following impairment of the protein quality control systems. During the disease course, these mutated proteins and aberrant peptides are released in the extracellular milieu as soluble or aggregated forms through a variety of mechanisms. Internalization by recipient cells may seed further aggregation and amplify existing proteostatic imbalances, thus triggering a vicious cycle that propagates pathology in vulnerable cells, such as motor neurons and other susceptible neuronal subtypes. Here, we provide an in‐depth review of ALS pathology with a particular focus on the disease mechanisms of seeding and transmission of the most common ALS‐associated proteins, including SOD1, FUS, TDP‐43, and C9orf72‐linked dipeptide repeats. For each of these proteins, we report historical, biochemical, and pathological evidence of their behaviors in ALS. We further discuss the possibility to harness pathological proteins as biomarkers and reflect on the implications of these findings for future research.     

FUS/TLS forms cytoplasmic aggregates,inhibits cell growth and interacts with TDP-43 in a yeast model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by the premature loss of motor neurons. While the underlying cellular mechanisms of neuron degeneration are unknown, the cytoplasmic aggregation of several proteins is associated with sporadic and familial forms of the disease. Both wild-type and mutant forms of the RNA-binding proteins FUS and TDP-43 accumulate in cytoplasmic inclusions in the neurons of ALS patients. It is not known if these so-called proteinopathies are due to a loss of function or a gain of toxicity resulting from the formation of cytoplasmic aggregates. Here we present a model of FUS toxicity using the yeast Saccharomyces cerevisiae in which toxicity is associated with greater expression and accumulation of FUS in cytoplasmic aggregates. We find that FUS and TDP-43 have a high propensity for co-aggregation, unlike the aggregation patterns of several other aggregation-prone proteins. Moreover, the biophysical properties of FUS aggregates in yeast are distinctly different from many amyloidogenic proteins, suggesting they are not composed of amyloid.     

RNA-binding proteins with prion-like domains in ALS and FTLD-U

Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig''s disease) is a debilitating and universally fatal neurodegenerative disease that devastates upper and lower motor neurons. The causes of ALS are poorly understood. A central role for RNA-binding proteins and RNA metabolism in ALS has recently emerged. The RNA-binding proteins TDP-43 and FUS are principal components of cytoplasmic inclusions found in motor neurons of ALS patients and mutations in TDP-43 and FUS are linked to familial and sporadic ALS. Pathology and genetics also connect TDP-43 and FUS with frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). It was unknown whether mechanisms of FUS aggregation and toxicity were similar or different to those of TDP-43. To address this issue, we have employed yeast models and pure protein biochemistry to define mechanisms underlying TDP-43 and FUS aggregation and toxicity, and to identify genetic modifiers relevant to human disease. We have identified prion-like domains in FUS and TDP-43 and provide evidence that these domains are required for aggregation. Our studies have defined key similarities as well as important differences between the two proteins. Collectively, our findings lead us to suggest that FUS and TDP-43, though similar RNA-binding proteins, likely aggregate and confer disease phenotypes via distinct mechanisms.Key words: TDP-43, FUS/TLS, yeast, ALS, FTLD-U, prion     

A role of small heat shock protein B8 (HspB8) in the autophagic removal of misfolded proteins responsible for neurodegenerative diseases

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motorneurons. As with other age-dependent neurodegenerative disorders, ALS is linked to the presence of misfolded proteins that may perturb several intracellular mechanisms and trigger neurotoxicity. Misfolded proteins aggregate intracellularly generating insoluble inclusions that are a key neuropathological hallmark of ALS. Proteins involved in the intracellular degradative systems, signalling pathways and the human TAR DNA-binding protein TDP-43 are major components of these inclusions. While their role and cytotoxicity are still largely debated, aggregates represent a powerful marker to follow protein misfolding in the neurodegenerative processes. Using in vitro and in vivo models of mutant SOD1 associated familial ALS (fALS), we and other groups demonstrated that protein misfolding perturbs one of the major intracellular degradative pathways, the ubiquitin proteasome system, giving rise to a vicious cycle that leads to the further deposit of insoluble proteins and finally to the formation of inclusions. The aberrant response to mutated SOD1 thus leads to the activation of the cascade of events ultimately responsible for cell death. Hence, our idea is that, by assisting protein folding, we might reduce protein aggregation, restore a fully functional proteasome activity and/or other cascades of events triggered by the mutant proteins responsible for motorneuron death in ALS. This could be obtained by stimulating mutant protein turnover, using an alternative degradative pathway that could clear mutant SOD1, namely autophagy.     

Prion‐promoted phosphorylation of heterologous amyloid is coupled with ubiquitin‐proteasome system inhibition and toxicity

Many neurodegenerative diseases are associated with conversion of a soluble protein into amyloid deposits, but how this is connected to toxicity remains largely unknown. Here, we explore mechanisms of amyloid associated toxicity using yeast. [PIN⁺], the prion form of the Q/N‐rich Rnq1 protein, was known to enhance aggregation of heterologous proteins, including the overexpressed Q/N‐rich amyloid forming domain of Pin4 (Pin4C), and Pin4C aggregates were known to attract chaperones, including Sis1. Here we show that in [PIN⁺] but not [pin^?] cells, overexpression of Pin4C is deadly and linked to hyperphosphorylation of aggregated Pin4C. Furthermore, Pin4C aggregation, hyperphosphorylation and toxicity are simultaneously reversed by Sis1 overexpression. Toxicity may result from proteasome overload because hyperphosphorylated Pin4C aggregation is associated with reduced degradation of a ubiquitin‐protein degradation reporter. Finally, hyperphosphorylation of endogenous full‐length Pin4 was also facilitated by [PIN⁺], revealing that a prion can regulate post‐translational modification of another protein.     

Engineering enhanced protein disaggregases for neurodegenerative disease

Abstract

Protein misfolding and aggregation underpin several fatal neurodegenerative diseases, including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). There are no treatments that directly antagonize the protein-misfolding events that cause these disorders. Agents that reverse protein misfolding and restore proteins to native form and function could simultaneously eliminate any deleterious loss-of-function or toxic gain-of-function caused by misfolded conformers. Moreover, a disruptive technology of this nature would eliminate self-templating conformers that spread pathology and catalyze formation of toxic, soluble oligomers. Here, we highlight our efforts to engineer Hsp104, a protein disaggregase from yeast, to more effectively disaggregate misfolded proteins connected with PD, ALS, and FTD. Remarkably subtle modifications of Hsp104 primary sequence yielded large gains in protective activity against deleterious α-synuclein, TDP-43, FUS, and TAF15 misfolding. Unusually, in many cases loss of amino acid identity at select positions in Hsp104 rather than specific mutation conferred a robust therapeutic gain-of-function. Nevertheless, the misfolding and toxicity of EWSR1, an RNA-binding protein with a prion-like domain linked to ALS and FTD, could not be buffered by potentiated Hsp104 variants, indicating that further amelioration of disaggregase activity or sharpening of substrate specificity is warranted. We suggest that neuroprotection is achievable for diverse neurodegenerative conditions via surprisingly subtle structural modifications of existing chaperones.     

Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition

Disruption of the functional protein balance in living cells activates protective quality control systems to repair damaged proteins or sequester potentially cytotoxic misfolded proteins into aggregates. The established model based on Saccharomyces cerevisiae indicates that aggregating proteins in the cytosol of eukaryotic cells partition between cytosolic juxtanuclear (JUNQ) and peripheral deposits. Substrate ubiquitination acts as the sorting principle determining JUNQ deposition and subsequent degradation. Here, we show that JUNQ unexpectedly resides inside the nucleus, defining a new intranuclear quality control compartment, INQ, for the deposition of both nuclear and cytosolic misfolded proteins, irrespective of ubiquitination. Deposition of misfolded cytosolic proteins at INQ involves chaperone‐assisted nuclear import via nuclear pores. The compartment‐specific aggregases, Btn2 (nuclear) and Hsp42 (cytosolic), direct protein deposition to nuclear INQ and cytosolic (CytoQ) sites, respectively. Intriguingly, Btn2 is transiently induced by both protein folding stress and DNA replication stress, with DNA surveillance proteins accumulating at INQ. Our data therefore reveal a bipartite, inter‐compartmental protein quality control system linked to DNA surveillance via INQ and Btn2.     

Oxidation-induced misfolding and aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis

The presence of intracellular aggregates that contain Cu/Zn superoxide dismutase (SOD1) in spinal cord motor neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although SOD1 is abundant in all cells, its half-life in motor neurons far exceeds that in any other cell type. On the basis of the premise that the long half-life of the protein increases the potential for oxidative damage, we investigated the effects of oxidation on misfolding/aggregation of SOD1 and ALS-associated SOD1 mutants. Zinc-deficient wild-type SOD1 and SOD1 mutants were extremely prone to form visible aggregates upon oxidation as compared with wild-type holo-protein. Oxidation of select histidine residues that bind metals in the active site mediates SOD1 aggregation. Our results provide a plausible model to explain the accumulation of SOD1 aggregates in motor neurons affected in ALS.     

Critical role of the proline-rich region in Huntingtin for aggregation and cytotoxicity in yeast

Nine neurodegenerative diseases, such as Huntington, are caused by a polyglutamine (poly(Q)) expansion in otherwise unrelated proteins. Although poly(Q) expansion causes aggregation of the affected proteins, the protein context might determine the selective neuronal vulnerability found in each disease. Here we have report that, although expression of Huntingtin derivatives with a pathological poly(Q) expansion are innocuous in yeast, deletion of the flanking proline-rich region alters the shape and number of poly(Q) inclusions and unmasks toxic properties. Strikingly, deletion of Hsp104 increases the size of inclusions formed by expanded poly(Q) lacking the proline-rich region and abolishes toxicity. Overexpression of the chaperones Hsp104 or Hsp70 rescues growth defects in affected cells without resolving inclusions. However, aggregates formed by nontoxic Huntingtin derivatives or by toxic derivatives cured by chaperones are physically distinct from aggregates formed by toxic proteins. This study identifies the proline-rich region in Huntingtin as a profound cis-acting modulator of expanded poly(Q) toxicity and distinguishes between aggregates of toxic or non-toxic proteins.     

Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates

Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein-sorting factors of the Hook family, termed Btn2 and Cur1, as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. On the basis of these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein-sorting factors. Our model suggests that protein aggregation is not a haphazard process but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.     

Curing of the [URE3] prion by Btn2p, a Batten disease-related protein

[URE3] is a prion (infectious protein), a self-propagating amyloid form of Ure2p, a regulator of yeast nitrogen catabolism. We find that overproduction of Btn2p, or its homologue Ypr158 (Cur1p), cures [URE3]. Btn2p is reported to be associated with late endosomes and to affect sorting of several proteins. We find that double deletion of BTN2 and CUR1 stabilizes [URE3] against curing by several agents, produces a remarkable increase in the proportion of strong [URE3] variants arising de novo and an increase in the number of [URE3] prion seeds. Thus, normal levels of Btn2p and Cur1p affect prion generation and propagation. Btn2p-green fluorescent protein (GFP) fusion proteins appear as a single dot located close to the nucleus and the vacuole. During the curing process, those cells having both Ure2p-GFP aggregates and Btn2p-RFP dots display striking colocalization. Btn2p curing requires cell division, and our results suggest that Btn2p is part of a system, reminiscent of the mammalian aggresome, that collects aggregates preventing their efficient distribution to progeny cells.     

Recruitment into stress granules prevents irreversible aggregation of FUS protein mislocalized to the cytoplasm

Fused in sarcoma (FUS) belongs to the group of RNA-binding proteins implicated as underlying factors in amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. Multiple FUS gene mutations have been linked to hereditary forms, and aggregation of FUS protein is believed to play an important role in pathogenesis of these diseases. In cultured cells, FUS variants with disease-associated amino acid substitutions or short deletions affecting nuclear localization signal (NLS) and causing cytoplasmic mislocalization can be sequestered into stress granules (SGs). We demonstrated that disruption of motifs responsible for RNA recognition and binding not only prevents SG recruitment, but also dramatically increases the protein propensity to aggregate in the cell cytoplasm with formation of juxtanuclear structures displaying typical features of aggresomes. Functional RNA-binding domains from TAR DNA-binding protein of 43 kDa (TDP-43) fused to highly aggregation-prone C-terminally truncated FUS protein restored the ability to enter SGs and prevented aggregation of the chimeric protein. Truncated FUS was also able to trap endogenous FUS molecules in the cytoplasmic aggregates. Our data indicate that RNA binding and recruitment to SGs protect cytoplasmic FUS from aggregation, and loss of this protection may trigger its pathological aggregation in vivo.