An Hsp90 co-chaperone links protein folding and degradation and is part of a conserved protein quality control

In this paper, we show that the essential Hsp90 co-chaperone Sgt1 is a member of a general protein quality control network that links folding and degradation through its participation in the degradation of misfolded proteins both in the cytosol and the endoplasmic reticulum (ER). Sgt1-dependent protein degradation acts in a parallel pathway to the ubiquitin ligase (E3) and ubiquitin chain elongase (E4), Hul5, and overproduction of Hul5 partly suppresses defects in cells with reduced Sgt1 activity. Upon proteostatic stress, Sgt1 accumu- lates transiently, in an Hsp90- and proteasome-dependent manner, with quality control sites (Q-bodies) of both yeast and human cells that co-localize with Vps13, a protein that creates organelle contact sites. Misfolding disease proteins, such as synphilin-1 involved in Parkinson's disease, are also sequestered to these compartments and require Sgt1 for their clearance

Using reporters of different misfolded proteins reveals differential strategies in processing protein aggregates

The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regardless of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7, gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, highlighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the protein disaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was not hindered when pro3-1 colocalized with a slowly resolved misfolded protein. Finally, we observed using super-resolution light microscopy as well as immunogold labeling EM in which both showed an even distribution of the different misfolded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compartmentalization, allows for differential clearance of these misfolding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing

Using reporters of different misfolded proteins reveals differential strategies in processing protein aggregates

The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regard less of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7,gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, high-lighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the proteindisaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was nothindered when pro3-1 colocalized with a slowly resolved mis-folded protein. Finally, we observed using super-resolutionlight microscopy as well as immunogold labeling EM in which both showed an even distribution of the different mis-folded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compart-mentalization, allows for differential clearance of these mis-folding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing

FMN reduces Amyloid-β toxicity in yeast by regulating redox status and cellular metabolism

Alzheimer\u27s disease (AD) is defined by progressive neurodegeneration, with oligomerization and aggregation of amyloid-β peptides (Aβ) playing a pivotal role in its pathogenesis. In recent years, the yeast Saccharomyces cerevisiae has been successfully used to clarify the roles of different human proteins involved in neurodegeneration. Here, we report a genome-wide synthetic genetic interaction array to identify toxicity modifiers of Aβ42, using yeast as the model organism. We find that FMN1, the gene encoding riboflavin kinase, and its metabolic product flavin mononucleotide (FMN) reduce Aβ42 toxicity. Classic experimental analyses combined with RNAseq show the effects of FMN supplementation to include reducing misfolded protein load, altering cellular metabolism, increasing NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios and increasing resistance to oxidative stress. Additionally, FMN supplementation modifies Htt103QP toxicity and α-synuclein toxicity in the humanized yeast. Our findings offer insights for reducing cytotoxicity of Aβ42, and potentially other misfolded proteins, via FMN-dependent cellular pathways

Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates and delays senescence

Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to maintaining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity

Komponenten und Mechanismen der cytoplasmatischen Proteinqualitätskontrolle und des Abbaus von regulatorischen Enzymen

Relatively little is known about cytoplasmic protein quality control in eukaryotic cells. After proteins have been translated on ribosomes, they have to achieve their native conformation, get to their place of action and be assembled into protein complexes when indicated. Errors in the protein sequence caused by DNA mutations, mistakes during transcription or translation, as well as folding disorders caused by chemical or physical stress can impair the proper functionality of the cell and evoke diseases. Therefore, it is the task of the cellular protein quality control system to assist proteins while folding into their native conformation, to unfold misfolded proteins and to refold them. Finally, irreversibly misfolded proteins have to be transferred for degradation to the proteolytic systems of the cell, the 26S proteasome or the vacuole (lysosome). The components that are involved in the control of protein folding and in the transfer of misfolded cytoplasmic proteins to the proteolytic systems have been poorly investigated. In this work, novel components of the cytoplasmic quality control system have been discovered by studying mutated variants of carboxypeptidase Y (CPY*), a vacuolar enzyme, which due to deletion of its signal sequence cannot be imported into the endoplasmic reticulum (ER) for further transfer into the vacuole and therefore is permanently located to the cytoplasm of the budding yeast Saccharomyces cerevisiae. Studies investigating ∆ssCPY* (signal sequence deleted CPY*), ∆ssCG* (∆ssCPY* carrying a C-terminal GFP tag) and the corresponding wild-type enzyme ∆ssCPY showed that for proteasomal degradation of these substrates the cytoplasmic chaperone Hsp70 (Heat shock protein) Ssa1, the Hsp40 co-chaperone Ydj1 and the ubiquitin-conjugating enzymes (E2) Ubc4 and Ubc5 are necessary. It could be shown that Ssa1 and Ydj1 are involved in the resolubilization of precipitated ∆ssCG*, in keeping ∆ssCG* in solution and in the transport of ubiquitylated ∆ssCG* to the 26S proteasome. The following study searched for further factors of the cytoplasmic quality control, especially a ubiquitin ligase (E3), which is capable of targeting misfolded cytoplasmic proteins for proteasomal degradation. Yeast mutants were isolated in a genetic screen, which are able to stabilize the fusion protein ∆ssCL*myc (∆ssCPY* C-terminally fused to myc-tagged 3-isopropylmalate dehydrogenase (LEU2myc)) and are therefore able to grow on media lacking leucine. This led to the discovery of the E3 Ubr1. Subsequent investigations revealed that the proteasomal degradation of ∆ssCL*myc is strongly dependent on Ubr1 and that the misfolded substrate physically interacts with this E3. Furthermore, it could be shown that for degradation of ∆ssCL*myc and ∆ssCG* the Hsp110s Sse1 and Sse2 are necessary, probably functioning as nucleotide exchange factors for Ssa1. Besides the degradation of finally misfolded cytoplasmic proteins, the eukaryotic cell utilizes its proteolytic systems to eliminate regulatory enzymes upon changes in the cellular environment. After switching cells from non-fermentable to fermentable media, a key regulatory enzyme in the gluconeogenesis pathway, fructose-1,6-bisphosphatase (FBPase), is ubiquitylated by the Gid-E3 complex and then degraded by the ubiquitin proteasome system (UPS) to allow switching from gluconeogenesis to glycolysis. In a further study we found that for degradation of ubiquitylated FBPase procession by the AAA-ATPase Cdc48 and its co-factors Ufd1 and Npl4 is necessary. This is the first time that for degradation of a native substrate by the UPS a dependency on the Cdc48-Ufd1-Npl4 complex could be shown. In addition, it could be shown that the ubiquitin receptor proteins Dsk2 and Rad23 are also necessary for the proteasomal degradation of FBPase. Before a ubiquitylated substrate of the 26S proteasome is degraded, its ubiquitin chains are cleaved off. The ubiquitin-specific protease Ubp14 cleaves these free chains to single ubiquitin molecules. Cells deleted in UBP14 accumulate ubiquitin chains, which leads to impairment of the UPS dependent protein degradation. In a further study we demonstrated that inhibition of proteasomal degradation by deletion of UBP14 does not occur in the degradation process of all substrates tested. While e.g. UPS dependent degradation of the gluconeogenic enzyme FBPase is impaired in ∆ubp14 strains, degradation of ∆ssCG* is only slightly reduced and degradation of a misfolded substrate of the ER, CPY*HA is not at all affected. This finding suggests that there are several substrate specific pathways to proteasomal degradation, which can be defined by a varying dependency on Ubp14.Über die Proteinqualitätskontrolle im Zytoplasma von eukaryontischen Zellen ist vergleichsweise wenig bekannt. Nachdem Proteine an den Ribosomen translatiert wurden, müssen sie sich in ihre native Konformation falten und an ihren Wirkungsort gelangen. Fehler in der Proteinsequenz, verursacht durch Mutationen der DNA, Transkriptions- oder Translationsfehler, sowie durch chemischen und physikalischen Stress auftretende Faltungsstörungen der Proteine, können die korrekte Funktionsweise der Zelle stören und Krankheiten hervorrufen. Aufgabe der Proteinqualitätskontrolle der Zelle ist es daher, Proteinen bei der Faltung in ihre natürliche Konformation zu helfen, fehlgefaltete Proteine zu entfalten und wieder von neuem zu falten. Endgültig fehlgefaltete Proteine müssen den proteolytischen Systemen der Zelle, 26S Proteasom oder Vakuole (Lysosom), zum Abbau zugeführt werden. Über die Komponenten, welche im Zytoplasma an der Durchführung und Kontrolle der korrekten Faltung, sowie an der Übergabe an die proteolytischen Systeme beteiligt sind, ist relativ wenig bekannt. Mittels Studien an mutierten Varianten des vakuolären Enzyms Carboxypeptidase Y (CPY*), die aufgrund genetischer Entfernung ihrer Signalsequenzen nicht in das Endoplasmatische Retikulum (ER) zum Weitertransport in die Vakuole importiert werden können, und daher permanent im Zytoplasma der Knospungs-Hefe Saccharomyces cerevisiae verbleiben, wurden neue Komponenten der zytoplasmatischen Qualitätskontrolle entdeckt. Durch Studien an ∆ssCPY* (signalsequenzdeletierte CPY*), ∆ssCG* (∆ssCPY* mit C-terminalem GFP), sowie am entsprechenden Wildtypenzym ∆ssCPY konnte gezeigt werden, dass für den proteasomalen Abbau das zytoplasmatische Hsp70 (Hitzeschockprotein) Ssa1, das Hsp40 Ko-Chaperon Ydj1, sowie die ubiquitin-konjugierenden Enzyme (E2) Ubc4 und Ubc5 notwendig sind. Es konnte gezeigt werden, dass Ssa1 und Ydj1 an der Wiederauflösung von ausgefallenem ∆ssCG*, an dem Prozess es in Lösung zu halten und am Transport von ubiquitinyliertem ∆ssCG* zum 26S Proteasom beteiligt sind. In einer anschließenden Studie wurde nach weiteren Faktoren der zytoplasmatischen Qualitätskontrolle gesucht, insbesondere nach einer Ubiquitinligase (E3), welche in der Lage ist, fehlgefaltete zytoplasmatische Proteine durch spezifische Ubiquitinylierung dem proteasomalen Abbau zu übergeben. Dafür wurden in einem genetischen Screen Hefemutanten isoliert, welche das Fusionsprotein ∆ssCL*myc (∆ssCPY* mit C-terminaler Myc getaggter 3-Isopropylmalatdehydrogenase (LEU2myc)) stabilisieren und dadurch auf Medium ohne Leucin wachsen können. Dabei wurde das E3 Ubr1 gefunden. Durch anschließende Untersuchungen konnte gezeigt werden, dass der proteasomale Abbau von ∆ssCL*myc stark von Ubr1 abhängig ist, sowie der Befund erhoben werden, dass das fehlgefaltete Substrat mit diesem E3 physikalisch interagiert. Ferner konnte gezeigt werden, dass für den Abbau von ∆ssCL*myc und ∆ssCG* die Hsp110 Proteine Sse1 und Sse2, wahrscheinlich in ihrer Funktion als Nukleotidaustauschfaktoren für Ssa1, notwendig sind. Neben endgültig fehlgefalteten zytoplasmatischen Proteinen, entfernt das Ubiquitin Proteasom System (UPS) der eukaryontischen Zelle auch regulatorische Enzyme bei sich verändernden Umweltbedingungen. Werden Zellen von einem nicht-fermentierbarem auf fermentierbares Medium gewechselt, wird ein Schlüsselenzym der Gluconeogenese, Fructose-1,6-bisphosphatase (FBPase), durch den Gid-E3-Komplex ubiquitinyliert und dann durch das UPS abgebaut, um von Gluconeogenese auf Glycolyse umzuschalten. In einer weiteren Studie konnte gezeigt werden, dass für den Abbau von ubiquitinylierter FBPase die Weiterverarbeitung durch die AAA-ATPase Cdc48 und seine Kofaktoren Ufd1 und Npl4 notwendig ist. Damit konnte zum ersten mal für ein natürliches Substrat des UPS eine Abhängigkeit vom Cdc48-Ufd1-Npl4 Komplex gezeigt werden. Auch konnte gezeigt werden, dass die Ubiquitinrezeptorproteine Dsk2 und Rad23 für den proteasomalen Abbau der FBPase notwendig sind. Vor dem Abbau eines ubiquitinylierten Substrates durch das Proteasom werden die Ubiquitinketten abgeschnitten. Die ubiquitin-spezifische Protease Ubp14 spaltet dann diese freien Ketten zu monomeren Ubiquitineinheiten. In UBP14 deletierten Hefestämmen akkumulieren Ubiquitinketten, was dazu führt, dass der gesamte UPS abhängige Proteinabbau gestört wird. In einer weiteren Studie konnte gezeigt werden, dass die Hemmung des proteasomalen Abbaus durch Deletion von UBP14 nicht, wie ursprünglich angenommen, für alle Substrate des UPS gleichermaßen gilt. Während z.B. der UPS abhängige Abbau des gluconeogenetischen Enzyms FBPase in ∆ubp14 Stämmen gehemmt ist, ist der Abbau von ∆ssCG* nur wenig und der von einem fehlgefalteten Substrat des Endoplasmatischen Retikulums (ER), CPY*HA, überhaupt nicht gestört. Dieser Befund deutet darauf hin, dass es verschiedene substratspezifische Wege zum Proteasom gibt, welche sich durch eine variierende Abhängigkeit von Ubp14 beschreiben lassen

Differential role of cytosolic Hsp70s in longevity assurance and protein quality control

70 kDa heat shock proteins (Hsp70) are essential chaperones of the protein quality control network; vital for cellular fitness and longevity. The four cytosolic Hsp70's in yeast, Ssa1-4, are thought to be functionally redundant but the absence of Ssa1 and Ssa2 causes a severe reduction in cellular reproduction and accelerates replicative aging. In our efforts to identify which Hsp70 activities are most important for longevity assurance, we systematically investigated the capacity of Ssa4 to carry out the different activities performed by Ssa1/2 by overproducing Ssa4 in cells lacking these Hsp70 chaperones. We found that Ssa4, when overproduced in cells lacking Ssa1/2, rescued growth, mitigated aggregate formation, restored spatial deposition of aggregates into protein inclusions, and promoted protein degradation. In contrast, Ssa4 overproduction in the Hsp70 deficient cells failed to restore the recruitment of the disaggregase Hsp104 to misfolded/aggregated proteins, to fully restore clearance of protein aggregates, and to bring back the formation of the nucleolus-associated aggregation compartment. Exchanging the nucleotide-binding domain of Ssa4 with that of Ssa1 suppressed this 'defect' of Ssa4. Interestingly, Ssa4 overproduction extended the short lifespan of ssa1 Delta ssa2 Delta mutant cells to a lifespan comparable to, or even longer than, wild type cells, demonstrating that Hsp104-dependent aggregate clearance is not a prerequisite for longevity assurance in yeast. Author summary: All organisms have proteins that network together to stabilize and protect the cell throughout its lifetime. One of these types of proteins are the Hsp70s (heat shock protein 70). Hsp70 proteins take part in folding other proteins to their functional form, untangling proteins from aggregates, organize aggregates inside the cell and ensure that damaged proteins are destroyed. In this study, we investigated three closely related Hsp70 proteins in yeast; Ssa1, 2 and 4, in an effort to describe the functional difference of Ssa4 compared to Ssa1 and 2 and to answer the question: What types of cellular stress protection are necessary to reach a normal lifespan? We show that Ssa4 can perform many of the same tasks as Ssa1 and 2, but Ssa4 doesn't interact in the same manner as Ssa1 and 2 with other types of proteins. This leads to a delay in removing protein aggregates created after heat stress. Ssa4 also cannot ensure that misfolded proteins aggregate correctly inside the nucleus of the cell. However, this turns out not to be necessary for yeast cells to achieve a full lifespan, which shows us that as long as cells can prevent aggregates from forming in the first place, they can reach a full lifespan

MRI monitoring of pathological changes in the spinal cord in patients with multiple sclerosis

The spinal cord is a clinically important site that is affected by pathological changes in most patients with multiple sclerosis; however, imaging of the spinal cord with conventional MRI can be difficult. Improvements in MRI provide a major advantage for spinal cord imaging, with better signal-to-noise ratio and improved spatial resolution. Through the use of multiplanar MRI, identification of diffuse and focal changes in the whole spinal cord is now routinely possible. Corroborated by related histopathological analyses, several new techniques, such as magnetisation transfer, diffusion tension imaging, functional MRI, and proton magnetic resonance spectroscopy, can detect non-focal, spinal cord pathological changes in patients with multiple sclerosis. Additionally, functional MRI can reveal changes in the response pattern to sensory stimulation in patients with multiple sclerosis. Through use of these techniques, findings of cord atrophy, intrinsic cord damage, and adaptation are shown to occur largely independently of focal spinal cord lesion load, which emphasises their relevance in depiction of the true burden of disease. Combinations of magnetisation transfer ratio or diffusion tension imaging indices with cord atrophy markers seem to be the most robust and meaningful biomarkers to monitor disease evolution in early multiple sclerosis