Functional Modules of the Proteostasis Network

Cells invest in an extensive network of factors to maintain protein homeostasis (proteostasis) and prevent the accumulation of potentially toxic protein aggregates. This proteostasis network (PN) comprises the machineries for the biogenesis, folding, conformational maintenance, and degradation of proteins with molecular chaperones as central coordinators. Here, we review recent progress in understanding the modular architecture of the PN in mammalian cells and how it is modified during cell differentiation. We discuss the capacity and limitations of the PN in maintaining proteome integrity in the face of proteotoxic stresses, such as aggregate formation in neurodegenerative diseases. Finally, we outline various pharmacological interventions to ameliorate proteostasis imbalance

Proteasome dysfunction induces excessive proteome instability and loss of mitostasis that can be mitigated by enhancing mitochondrial fusion or autophagy

The ubiquitin-proteasome pathway (UPP) is central to proteostasis network (PN) functionality and proteome quality control. Yet, the functional implication of the UPP in tissue homeodynamics at the whole organism level and its potential cross-talk with other proteostatic or mitostatic modules are not well understood. We show here that knock down (KD) of proteasome subunits in Drosophila flies, induced, for most subunits, developmental lethality. Ubiquitous or tissue specific proteasome dysfunction triggered systemic proteome instability and activation of PN modules, including macroautophagy/autophagy, molecular chaperones and the antioxidant cncC (the fly ortholog of NFE2L2/Nrf2) pathway. Also, proteasome KD increased genomic instability, altered metabolic pathways and severely disrupted mitochondrial functionality, triggering a cncC-dependent upregulation of mitostatic genes and enhanced rates of mitophagy. Whereas, overexpression of key regulators of antioxidant responses (e.g., cncC or foxo) could not suppress the deleterious effects of proteasome dysfunction; these were alleviated in both larvae and adult flies by modulating mitochondrial dynamics towards increased fusion or by enhancing autophagy. Our findings reveal the extensive functional wiring of genomic, proteostatic and mitostatic modules in higher metazoans. Also, they support the notion that age-related increase of proteotoxic stress due to decreased UPP activity deregulates all aspects of cellular functionality being thus a driving force for most age-related diseases. Abbreviations: ALP: autophagy-lysosome pathway; ARE: antioxidant response element; Atg8a: autophagy-related 8a; ATPsynβ: ATP synthase, β subunit; C-L: caspase-like proteasomal activity; cncC: cap-n-collar isoform-C; CT-L: chymotrypsin-like proteasomal activity; Drp1: dynamin related protein 1; ER: endoplasmic reticulum; foxo: forkhead box, sub-group O; GLU: glucose; GFP: green fluorescent protein; GLY: glycogen; Hsf: heat shock factor; Hsp: Heat shock protein; Keap1: kelch-like ECH-associated protein 1; Marf: mitochondrial assembly regulatory factor; NFE2L2/Nrf2: nuclear factor, erythroid 2 like 2; Opa1: optic atrophy 1; PN: proteostasis network; RNAi: RNA interference; ROS: reactive oxygen species; ref(2)P: refractory to sigma P; SQSTM1: sequestosome 1; SdhA: succinate dehydrogenase, subunit A; T-L: trypsin-like proteasomal activity; TREH: trehalose; UAS: upstream activation sequence; Ub: ubiquitin; UPR: unfolded protein response; UPP: ubiquitin-proteasome pathway.</p

Identification And Characterization Of Mediators Of Toxicity Of Aβ Oligomers By Genome Wide Screening In <i>Caenorhabditis Elegans</i>

Soluble oligomers of the amyloid-β (Aβ) protein play a key role in the pathogenesis of Alzheimer’s disease (AD), although the underlying molecular mechanisms are poorly understood. In order to search for proteins involved in the formation and/or toxicity of Aβ oligomers, a transgenic C. elegans model of AD was used in which inducible expression of Aβ oligomers results in a complete paralysis; in these worms a genetic screen following chemical mutagenesis was applied to discover the genes involved in the Aβ-dependent paralysis (forward genetics). This analysis allowed identification of a mutated clone showing a complete lack of paralysis, despite this it not bear mutations in the Aβ coding region, and accumulates Aβ transcript and protein levels comparable to that of the non-mutated strain. This is the first in vivo model in which the expression of Aβ oligomers do not result in any toxic effect. The genome of the mutated worm was then sequenced and compared with that of the control strain to search for altered genes. Two genes, with no known function, were found to bear a stop codon mutation, likely resulting in the translation of an inactive protein. The rest of the mutations were missense mutations. Among them, point mutations were observed in some genes previously correlated with nematode lifespan and ageing. In C. elegans these biological processes are coordinated by the insulin/IGF-1-like signalling (IIS) pathway, which also regulates the response of the organism to toxic aggregated proteins. Thus, the activation of the IIS pathway was investigated in control and mutated worms. As expected, Aβ expression induced the up-regulation of two genes coding for small heat shock proteins (a class of chaperons known to be involved in AD) in the control strain, whereas these genes were actually down-regulated in the mutated strain. Since heat shock proteins are known to bind Aβ oligomers, these chaperons could directly mediate the formation of toxic amyloid species. Moreover, the results of the whole genome sequencing indicate that several proteins could act as potential novel mediators of Aβ toxicity and could open up new insights for research on age-related, neurodegenerative diseases

Identifying modifiers of age‐dependent protein aggregation in C. elegans

The misfolding of specific proteins and their accumulation in insoluble aggregates has long been recognized as a pathological hallmark of several neurodegenerative diseases. In recent years, widespread protein aggregation occurring during healthy aging has become a hot topic of research. However, to this date little is known about the regulation of this aggregation, the tissue‐specificity and the consequences in a disease context. This thesis answers several questions about different aspects of protein aggregation with aging and in disease. Notably, we analysed the solubility of RNA‐binding proteins that are important for the formation of stress granules (sgRBPs) in the nematode Caenorhabditis elegans (C. elegans). We showed the impact of sgRBP insolubility on organismal health and the importance of maintaining their solubility in long‐lived animals. We identified regulators of sgRBP aggregation. In addition, we showed that aggregation‐prone sgRBPs are highly prone to interact with other proteins and that this co‐localization can influence aggregation patterns or protein localization. Furthermore, we analysed the tissue‐specificity of the regulation of age‐related protein aggregation. Disruption of the protein‐quality control network has contrasting effects on protein aggregation in different tissues, surprisingly reducing age‐related protein aggregation in the pharyngeal muscle of C. elegans. Specifically, we showed that impaired proteinquality control prevented the accumulation of newly synthesized aggregation‐prone proteins. Additionally we demonstrated how screening approaches identifying mutations that influence disease‐associated phenotypes, like protein aggregation in C. elegans, can help prioritise variants found by whole exome sequencing in large cohorts of patients with Parkinson’s disease. To validate promising candidates found to be influencing protein aggregation in C. elegans, we have established a cell culture model of age‐related protein aggregation. In conclusion, these findings give important insights into mechanism and regulation of age‐related protein insolubility and highlight the importance of age‐related protein aggregation for neurodegenerative diseases

Understanding proteostasis dysregulation in phenylketonuria – a paradigm disease for inborn error of metabolism

Fenylketonuria (PKU) er en medfødt stoffskiftesykdom forårsaket av mutasjoner i genet for fenylalanin hydroksylase (PAH), og fører til ustabile og ofte feilfoldete enzymer med nedsatt evne til å metabolisere aminosyren fenylalanin (Phe). Den påfølgende opphopningen av Phe i blodet (hyperfenylalaninemi, HPA) kan nå nevrotoksiske nivåer, og uten behandling vil dette føre til alvorlige fysiske og psykiske funksjonshemninger, epilepsi og adferdsproblemer. Pasienter behandles derfor med en streng lav-Phe diett fra fødsel og anbefales å forbli på dietten livet ut. Den fenotypiske variasjonen er høy, da de fleste pasientene er heterozygoter, og mer enn 1200 PKU mutasjoner er registrert. Flere musemodeller for PKU (Enu1, Enu2 og Enu3) har vært tilgjengelige for PKU-forskning, og i dette arbeidet presenteres en ny musemodell som representerer en mutasjon med høy utbredelse i pasienter, Pah-R261Q. Det komplekse proteostase-nettverket omfatter translasjonsmaskineriet, molekylære chaperoner og cochaperoner, ubiquitin-proteasom systemet (UPS) og autofagimaskineriet. Proteostase-nettverket bistår og kontrollerer dannelsen, foldingen og nivåene av native proteiner, og er spesielt involvert i å motvirke akkumuleringen av feilfoldete og aggregerte proteiner. Mutert PAH har tidligere vist seg å danne små aggregater som er antatt å bli nedbrutt av UPS, men mye er fortsatt uvisst når det kommer til proteostase-reguleringen og nedbrytningsmekanismene for PAH og dets mutanter i pasienter. DNAJC12, en molekylær cochaperon i HSP40 familien, har nylig blitt identifisert som en spesifikk cochaperon for de tetrahydrobiopterin (BH4)-avhengige aromatiske aminosyre-hydroksylasene, proteinfamilien PAH tilhører, og er viktig for deres korrekte folding og vedlikehold. Vi studerte rollen DNAJC12 har i folding og nedbrytning av villtype (WT) og mutert PAH, både in vitro og in vivo. Våre resultater viste lavere nivåer av PAH og DNAJC12 i cellene som uttrykte PAH-mutanter, samt i Enu1 mus (homozygote for Pah varianten V106A) sammenlignet med WT mus. Mesteparten av det muterte PAH, men ikke WT-PAH, viste seg å være mono-ubiquitinert i leverlysater fra mus. I tillegg viste eksperimenter med co-immunopresipitering i leverlysater komplekse formasjoner mellom DNAJC12 og PAH (både WT og ubiquitinert mutant), og våre resultater underbygger at cochaperonen spiller en rolle både i folding av PAH og i prosesseringen av dens misfoldede ubiquitinerte former. Vi genererte og karakteriserte Pah-R261Q musemodellen som viste BH4-responderende HPA. Det muterte PAH var feilfoldet og presenterte amyloid aggregering i lever, assosiert med en toksisk "gain-of-function". Denne typen aggregering har ikke tidligere vært vist for HPA eller PKU mutasjoner, og ser ut til å assosieres med deregulering av proteostase, endret lipid metabolisme og oksidativt stress. Sammenlignet med Enu1 og WT, som viser lignende mRNA-nivåer av DNAJC12, hadde Pah-R261Q en oppregulering av cochaperonen, som er en videre bekreftelse av oksidativt stress i denne musen. Til forskjell fra de mindre PAH aggregatene i Enu1, ser de ubiquitinerte amyloid-lignende PAH-aggregatene ut til å være for store for nedbrytning av UPS, og aktiveringen av autofagisystemet ble indikert ved kolokaliseringen av muterte PAH aggregater og markører for autofagi. Mens PKU tradisjonelt har vært ansett som en "loss-of-function" sykdom har vårt arbeid vist at noen mutasjoner kan vise en toksisk "gain-of-function" grunnet store PAH aggregater, som muligens fører til oksidativt stress og ytterligere tilleggssykdommer. Aldring har blitt påvist å føre til nedgang i proteostasenettverkets effektivitet, forstyrrelser i den proteomiske balansen og økt oksidativt stress, i tillegg til forverring av alders-relaterte sykdommer. Vi forventet derfor at Pah-R261Q musene, med deres store PAH aggregater i lever og økt oksidativt stress, skulle ha en redusert generell helsestatus, økte tilleggssykdommer og muligens tidlig død når de ble gamle. Den aldrende Pah-R261Q musen hadde en økt serum-konsentrasjon av Phe sammenlignet med både WT og unge Pah-R261Q, men viste ingen tegn på nevrologiske problemer. Flere av biomarkør-konsentrasjonene som indikerte oksidativt stress i de unge Pah-R261Q musene viste en uventet normalisering mot WT nivåer i de gamle musene. Selv om biomarkør-konsentrasjonene var like i gamle Pah-R261Q og WT mus så viste interrelasjons-plot mellom par av biomarkører en distinkt forskjell, som kan indikere at en annen underliggende metabolsk mekanisme kan påvirke de muterte musene. Den lille forbedringen i motorfunksjon og forebyggingen av tidlig død og tilleggssykdommer kan muligens forklares med den sterke aktiveringen av autofagi fra tidlig alder i den muterte musen. For å konkludere har dette arbeidet avdekket ny kunnskap om aggregeringen og nedbrytningen av mutert PAH; involvering av DNAJC12, visualisering av små PAH aggregater i lever fra Enu1 mus, store amyloid-lignende aggregater i lever fra Pah-R261Q mus og aktivering av autofagi for å bryte ned de store aggregatene. I Pah-R261Q musen fant vi en uventet toksisk "gain-of-function" grunnet feilfoldet PAH. PKU har vært en modellsykdom for mange medfødte stoffskiftesykdommer, og våre funn forventes å bidra til bedre mekanistisk forståelse av fenotypiske variasjon i flere av disse arvelige sykdommene.Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene, resulting in unstable and often misfolded enzyme with reduced ability to metabolize the amino acid phenylalanine (Phe). The subsequent buildup of blood Phe (hyperphenylalaninemia, HPA) can reach neurotoxic levels, and if left untreated it will result in severe intellectual disability, motor deficits, epilepsy, and behavioral problems. Patients are therefore treated with a strict low-Phe diet from birth and are advised to stay on the diet throughout their life. The phenotypic variability is high, as most patients are heterozygotes, and more than 1200 PKU mutations are registered. Several mouse models for PKU (Enu1, Enu2 and Enu3) have been available for PKU investigations, and in this work we present a novel mouse model representing a mutation with high prevalence in patients, Pah-R261Q. The complex proteostasis network, including the translational machinery, molecular chaperones and cochaperones, the ubiquitin-proteasome system (UPS), and the autophagy machinery, manages and controls the folding and levels of native proteins, and it is especially involved in counteracting the accumulation of misfolded and aggregated proteins. Mutated PAH has previously been shown to form small aggregates that are presumed to be degraded by the UPS, but much is unknown about the proteostasis regulation and the degradation mechanisms of PAH and its mutants in patients. Recently, DNAJC12, a molecular cochaperone of the HSP40 family, has been identified as the specific cochaperone of the tetrahydrobiopterin (BH4) dependent aromatic amino acid hydroxylases, the protein family to which PAH belongs, and is important for their correct folding and maintenance. We studied the role of DNAJC12 in the folding and degradation of wild-type (WT) and mutant PAH, both in vitro and in vivo. Our results showed lower levels of PAH and DNAJC12 in cells expressing PAH mutants, as well as in Enu1 mice (homozygous for the Pah variant V106A) compared to WT mice. Most mutant PAH, but not WT-PAH, appeared mono-ubiquitinated in mouse liver lysates. Moreover, by co-immunoprecipitation experiments in liver lysates we probed the complex formation between DNAJC12 and PAH (both WT and ubiquitinated mutant), and our results support that the cochaperone plays a role both in the folding of PAH and in the processing of its misfolded ubiquitinated conformations. Further, we generated and characterized the Pah-R261Q mouse model, which presented BH4 responsive HPA. The mutant PAH was misfolded and displayed amyloid aggregation in liver, associated with toxic gain-of-function. This type of aggregation has so far been unknown for HPA or PKU mutations, where it appears associated with proteostasis dysregulation, altered lipid metabolism, and oxidative stress. Compared with Enu1 and WT, which show comparable mRNA expression of DNAJC12, Pah- R261Q presented an upregulation of the cochaperone, further confirming the oxidative stress in this mouse. Differently to the smaller PAH aggregates in Enu1, the ubiquitinated amyloid-like PAH aggregates appear too large for the UPS, and engagement of the autophagy system was indicated by colocalization of mutant PAH aggregates and autophagy markers. Whereas PKU has traditionally been considered a loss-of-function disorder, our work shows that some mutations may display a toxic gainof- function due to large PAH aggregates, possibly leading to oxidative stress and additional comorbidities. Aging has been shown to lead to a decline in proteostasis network efficiency, disturbance of the proteomic balance and increased oxidative stress, as well as a worsening of age-related diseases. We therefore expected that the Pah-R261Q mice, with their large PAH aggregates in liver and increased oxidative stress, would exhibit a reduced general health, increased comorbidities, and possibly early death when reaching old age. The aging Pah-R261Q mice did present an increased serum concentration of Phe compared to both old WT and young Pah-R261Q mice but did not show signs of neurological issues. Unexpectedly, several of the biomarker concentrations indicating oxidative stress in the young Pah-R261Q mice seemed to have normalized towards WT levels in the old mice. Although the biomarker concentrations were similar in old Pah- R261Q and WT mice, interrelation plots between pairs of biomarkers revealed distinct differences, suggesting that different underlying metabolic mechanisms may be at work in the mutant mice. The slightly improved motor function and prevention of premature death and comorbidities may be explained by the strong activation of autophagy in the mutant mice from a young age. To conclude, this work has revealed new knowledge about the aggregation and degradation of mutant PAH with the involvement of DNAJC12, visualization of small PAH aggregates in Enu1 mouse livers, large amyloid-like aggregates in Pah-R261Q mouse livers, and engagement of autophagy to degrade the large aggregates. In Pah- R261Q mice we found the unexpected toxic gain-of-function of PAH misfolding. As PKU has been a model disease for many inborn errors of metabolism, the present findings are expected to contribute to a better mechanistic understanding of the phenotypic variability in these genetic disorders.Doktorgradsavhandlin

Proteome Stability as a Key Factor of Genome Integrity

DNA damage is constantly produced by both endogenous and exogenous factors; DNA lesions then trigger the so-called DNA damaged response (DDR). This is a highly synchronized pathway that involves recognition, signaling and repair of the damage. Failure to eliminate DNA lesions is associated with genome instability, a driving force in tumorigenesis. Proteins carry out the vast majority of cellular functions and thus proteome quality control (PQC) is critical for the maintenance of cellular functionality. PQC is assured by the proteostasis network (PN), which under conditions of proteome instability address the triage decision of protein fold, hold, or degrade. Key components of the PN are the protein synthesis modules, the molecular chaperones and the two main degradation machineries, namely the autophagy-lysosome and the ubiquitin-proteasome pathways; also, part of the PN are a number of stress-responsive cellular sensors including (among others) heat shock factor 1 (Hsf1) and the nuclear factor erythroid 2-related factor 2 (Nrf2). Nevertheless, the lifestyle- and/or ageing-associated gradual accumulation of stressors results in increasingly damaged and unstable proteome due to accumulation of misfolded proteins and/or protein aggregates. This outcome may then increase genomic instability due to reduced fidelity in processes like DNA replication or repair leading to various age-related diseases including cancer. Herein, we review the role of proteostatic machineries in nuclear genome integrity and stability, as well as on DDR responses

Mitochondrial Homeostasis and Cellular Senescence

Cellular senescence refers to a stress response aiming to preserve cellular and, therefore, organismal homeostasis. Importantly, deregulation of mitochondrial homeostatic mechanisms, manifested as impaired mitochondrial biogenesis, metabolism and dynamics, has emerged as a hallmark of cellular senescence. On the other hand, impaired mitostasis has been suggested to induce cellular senescence. This review aims to provide an overview of homeostatic mechanisms operating within mitochondria and a comprehensive insight into the interplay between cellular senescence and mitochondrial dysfunction