Chaperones, protein homeostasis & protein aggregation diseases

Eiwitten zijn de werkzame moleculen van cellen en moeten in een specifieke driedimensionale structuur zijn gevouwen. Niet gevouwen eiwitten zijn niet alleen niet-functioneel, maar kunnen samenklonteren en aggregaten vormen die ziektes kunnen veroorzaken (bijv. ziekte van Parkinson en ziekte van Huntington). Om dit tegen te gaan, hebben cellen een specifieke subgroep van eiwitten genaamd Heat Shock Proteins (HSPs). Zij voorkomen de vorming van aggregaten in een beschermend netwerk genaamd protein quality control network. Dit proefschrift beschrijft hoe onderdelen van het protein quality control network functioneren en welke rol deze spelen in humane ziektes. Verkeerd gevowuen eiwit moeten worden afgebroken. Wij hebben gevonden dat BAG-eiwitten, als regulatoren van Hsp70 dat de afwijkende eiwitten herkent, “beslissen” welk systeem gebruikt wordt voor eiwitafbraak. BAG1 stuurt Hsp70 aan tot het afleveren van zijn cliënten aan proteasomen, die oplosbare eiwitten afbreken. Als dit systeem niet werkt, maken cellen meer BAG3, dat ervoor zorgt dat Hsp70 zijn afwijkende eiwitten aflevert aan autofagosomen, waarmee eiwit aggregaten afgebroken kunnen worden. Mutaties in BAG3 veroorzaken erfelijke hartspierziektes omdat ze deze functie van BAG3 aantasten. Voor een erfelijke vorm van de ziekte van Parkinson hebben wij een subgroep van kleine HSPs (de HSPB familie) geïdentificeerd die bescherming kunnen bieden tegen de vorming van toxische aggregaten. Voor zowel de werking van de BAGs als van HSPBs is het niet nodig dat Hsp70 niveaus worden veranderd. Dit is belangrijk omdat een te hoog ,Hsp70 niveau de kans op kanker verhoogt. Therapie gericht op BAG3 of HSPBs zou dus mogelijk gebruikt kunnen worden in eiwit aggregatie ziektes.Proteins are the functional molecules of cells and have to be folded into a specific three-dimensional structure. Besides being non-functional, non-folded proteins can easily clump together and form aggregates that can cause disease (e.g. Parkinson’s disease and Huntington’s disease). To avoid this, cells contain special sets of proteins called Heat Shock Proteins (HSPs) that prevent aggregates formation in a defence network called the protein quality control network. This thesis is about how this protein quality control network functions and plays a role in human diseases.Misfolded proteins that can no longer be (re)folded, require degradation. We found that BAG proteins as regulators of Hsp70 which recognise the aberrant proteins, “decide” which system to use for protein degradation. For instance, BAG1 directs Hsp70 to deliver its clients to proteasomes that degrades soluble proteins. When this system is impaired, cells make more BAG3 that now directs Hsp70 to deliver the aberrant proteins to autophagosomes that also degrades protein aggregates. Interestingly, genetic mutations in BAG3 that cause heritable heart muscle diseases impair this BAG3 function, explaining why this mutation causes disease. For a heritable form of Parkinson’s disease, we identified a subset of small HSPs (called HSPB family) that can protect against the formation of toxic aggregates. Both the actions of BAGs and HSPBs do not require that Hsp70 levels have to be altered, which is of importance as elevating Hsp70 levels can increase the risk for cancer. Targeting BAG3 or HSPBs may thus be of potential usage in protein aggregation diseases

Barcoding heat shock proteins to human diseases: looking beyond the heat shock response

There are numerous human diseases that are associated with protein misfolding and the formation of toxic protein aggregates. Activating the heat shock response (HSR)--and thus generally restoring the disturbed protein homeostasis associated with such diseases--has often been suggested as a therapeutic strategy. However, most data on activating the HSR or its downstream targets in mouse models of diseases associated with aggregate formation have been rather disappointing. The human chaperonome consists of many more heat shock proteins (HSPs) that are not regulated by the HSR, however, and researchers are now focusing on these as potential therapeutic targets. In this Review, we summarize the existing literature on a set of aggregation diseases and propose that each of them can be characterized or 'barcoded' by a different set of HSPs that can rescue specific types of aggregation. Some of these 'non-canonical' HSPs have demonstrated effectiveness in vivo, in mouse models of protein-aggregation disease. Interestingly, several of these HSPs also cause diseases when mutated--so-called chaperonopathies--which are also discussed in this Review

Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks

BAG3 is a multi-domain hub that connects two classes of chaperones, small heat shock proteins (sHSPs) via two isoleucine-proline-valine (IPV) motifs and Hsp70 via a BAG domain.\ua0Mutations in either the IPV or BAG domain of BAG3 cause a dominant form of myopathy, characterized by protein aggregation in both skeletal and cardiac muscle tissues. Surprisingly, for both disease mutants, impaired chaperone binding is not sufficient to explain disease phenotypes. Recombinant mutants are correctly folded, show unaffected Hsp70 binding but are impaired in stimulating Hsp70-dependent client processing. As a consequence, the mutant BAG3 proteins become the node for a dominant gain of function causing aggregation of itself, Hsp70, Hsp70 clients and tiered interactors within the BAG3 interactome. Importantly, genetic and pharmaceutical interference with Hsp70 binding completely reverses stress-induced protein aggregation for both BAG3 mutations. Thus, the gain of function effects of BAG3 mutants act as Achilles heel of the HSP70 machinery

Chaperones, protein homeostasis & protein aggregation diseases

Eiwitten zijn de werkzame moleculen van cellen en moeten in een specifieke driedimensionale structuur zijn gevouwen. Niet gevouwen eiwitten zijn niet alleen niet-functioneel, maar kunnen samenklonteren en aggregaten vormen die ziektes kunnen veroorzaken (bijv. ziekte van Parkinson en ziekte van Huntington). Om dit tegen te gaan, hebben cellen een specifieke subgroep van eiwitten genaamd Heat Shock Proteins (HSPs). Zij voorkomen de vorming van aggregaten in een beschermend netwerk genaamd protein quality control network. Dit proefschrift beschrijft hoe onderdelen van het protein quality control network functioneren en welke rol deze spelen in humane ziektes. Verkeerd gevowuen eiwit moeten worden afgebroken. Wij hebben gevonden dat BAG-eiwitten, als regulatoren van Hsp70 dat de afwijkende eiwitten herkent, “beslissen” welk systeem gebruikt wordt voor eiwitafbraak. BAG1 stuurt Hsp70 aan tot het afleveren van zijn cliënten aan proteasomen, die oplosbare eiwitten afbreken. Als dit systeem niet werkt, maken cellen meer BAG3, dat ervoor zorgt dat Hsp70 zijn afwijkende eiwitten aflevert aan autofagosomen, waarmee eiwit aggregaten afgebroken kunnen worden. Mutaties in BAG3 veroorzaken erfelijke hartspierziektes omdat ze deze functie van BAG3 aantasten. Voor een erfelijke vorm van de ziekte van Parkinson hebben wij een subgroep van kleine HSPs (de HSPB familie) geïdentificeerd die bescherming kunnen bieden tegen de vorming van toxische aggregaten. Voor zowel de werking van de BAGs als van HSPBs is het niet nodig dat Hsp70 niveaus worden veranderd. Dit is belangrijk omdat een te hoog ,Hsp70 niveau de kans op kanker verhoogt. Therapie gericht op BAG3 of HSPBs zou dus mogelijk gebruikt kunnen worden in eiwit aggregatie ziektes

The family of mammalian small heat shock proteins (HSPBs):Implications in protein deposit diseases and motor neuropathies

A number of neurological and muscular disorders are characterized by the accumulation of aggregate-prone proteins and are referred to as protein deposit or protein conformation diseases. Besides some sporadic forms, most of them are genetically inherited in an autosomal dominant manner, although recessive forms also exist. Although genetically very heterogeneous, some of these diseases are the result of mutations in some members of the mammalian small heat shock protein family (sHSP/HSPB), which are key players of the protein quality control system and participate, together with other molecular chaperones and co-chaperones, in the maintenance of protein homeostasis. Thus, on one hand upregulation of specific members of the HSPB family can exert protective effects in protein deposit diseases, such as the polyglutamine diseases. On the other hand, mutations in the HSPBs lead to neurological and muscular disorders, which may be due to a loss-of-function in protein quality control and/or to a gain-of-toxic function, resulting from the aggregation-proneness of the mutants. In this review we summarize the current knowledge about some of the best characterized functions of the HSPBs (e.g. role in cytoskeleton stabilization, chaperone function, anti-aggregation and anti-apoptotic activities), also highlighting differences in the properties of the various HSPBs and how these may counteract protein aggregation diseases. We also describe the mutations in the various HSPBs associated with neurological and muscular disorders and we discuss how gain-of-toxic function mechanisms (e.g. due to the mutated HSPB protein instability and aggregation) and/or loss-of-function mechanisms can contribute to HSPB-associated pathologies. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology

HSPA1A-Independent Suppression of PARK2 C289G Protein Aggregation by Human Small Heat Shock Proteins

The C289G mutation of the parkin E3-ubiquitin protein ligase (PARK2) is associated with autosomal recessive juvenile onset Parkinson's disease and was found to be associated with protein aggregation. Members of the human small heat shock proteins (HSPBs) have been implicated in protein degradation and prevention of protein aggregation. In this study, we show that of the 10 HSPB members, individual overexpression of HSPB1, HSPB2, HSPB4, and HSPB7 suppresses PARK2 C289G-associated protein aggregation. Intriguingly, the protective actions of these HSPBs are not impaired upon inactivation of the ATP-dependent HSP70 chaperone machines. Depending on the HSPB member the protective actions involve either autophagic or proteasomal degradation pathways

Characterization of the myopathy associated BAG3 P209L mutation

Myofibrillar myopathy is a protein aggregate myopathy characterized by disintegration of the Z-disk and accumulation of protein aggregates. A dominant missense mutation (P209L) in the co-chaperone BAG3 has been associated with a severe early onset form of myofibrillar myopathy. The disease causing P209L mutation in BAG3 is located in it’s binding site for HSPB8 (IPV domain), suggesting it might alter a functional HSPB8-BAG3 interaction. Recently we showed that wildtype BAG3, in association with HSPB8 and HSP70, participates in prevention of misfolded protein aggregation through macro-autophagy. In cultured HEK293T-cells the BAG3 P209L mutant was found to be impaired in its ability to prevent polyQ aggregation. This, however occurs without major effects on binding to the BAG3-partners HSPB8 and HSP70. Just like wildtype BAG3, the BAG3 P209L mutant was able to enhance LC3 lipidation, suggesting that its effect on autophagy stimulation is not impaired. This was confirmed by the absence of direct effects of mutant BAG3 on autophagic flux, instead the mutant strikingly showed impairment in cargo-delivery. Our studies in Drosophila Melanogaster further substantiate the loss of function phenotype of BAG3 P209L as the main mechanism underlying BAG3 P209L-related myopathy

SENSING AND REROUTING OF PROTEIN DEGRADATION TOWARDS AUTOPHAGY UPON PROTEASOMAL IMPAIRMENT

The accumulation of misfolded, mutant proteins is a common basis for many adult onset neurodegenerative diseases. Cells have evolved an elaborate protein quality control system, which acts to facilitate the folding or refolding of misfolded protein species by molecular chaperones or, if folding is unsuccessful, these same chaperones often target the misfolded proteins for degradation, thereby preventing protein aggregation. Intracellular degradation is primarily mediated by two proteolytic systems: the autophagy and the ubiquitin proteasomal systems. Proteotoxic stress can lead to proteasomal impairment and augmented authophagosomal capacity in order to ensure proper clearance of clients (proteasome-autophagy switch). However, neither the mechanism of sensing nor that of switching is understood. Here, we show that the ER is main sensor for proteasomal inhibition through the IRE-1alpha-Xbp-1 signalling cascade. After proteasome inhibition, BAG-3 is upregulated in a HSF-1 independent manner, but in a Xbp-1 dependent manner and is a major executor of the proteasome-autophagy switch. BAG-3 both boosts autophagy and redirects HSP70-bound proteasomal clients to autophagosomes through competitive inhibition with its family member BAG-1, that normally directs HSP70-bound clients to the proteasome, thus playing a key role in the maintenance of protein homeostasis under proteotoxic stress conditions

BAG3 induces the sequestration of ubiquitinated proteins into cytoplasmic puncta and re-routes them to autophagy upon proteasomal impairment

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). As BAG3, a partner of the heat shock proteins HSPB8 and Hsp70, stimulates autophagy and its levels increase with aging, a condition characterized by decreased proteasome function and autophagy activation, it is tempting to speculate that BAG3 is required to re-route ubiquitinated clients to autophagy. Here, we show that BAG3 interacts via its BAG domain with ubiquitinated proteins and induces their sequestration into cytoplasmic puncta. Similarly, BAG3 drives, in an Hsp70-dependent manner, the recruitment of the proteasome client Ub-R-GFP into similar cytoplasmic puncta. These cytoplasmic puncta are co-labelled with canonical autophagy markers and linkers, suggesting that proteasomal client are re-routed to autophagy by BAG3. Indeed, upon proteasome inhibition ubiquitinated (insoluble) proteins accumulate in control cells, whilst in cells overexpressing BAG3 they are efficiently re-routed to autophagy for clearance. This action might be independent of HSPB8, which we find to dissociate from BAG3 early after proteasomal inhibition. Rather, HSPB8 becomes associated with RNA-containing stress granules, likely participating in translational arrest under proteasomal stress. Upon prolonged proteasomal inhibition, HSPB8 is then also massively recruited to the BAG3-positive puncta, tentatively to contribute to autophagy-mediated protein degradation of (other) accumulating misfolded substrates