Divergent genetic control of protein solubility and conformational quality in Escherichia coli

In bacteria, protein overproduction results in the formation of inclusion bodies, sized protein aggregates showing amyloid-like properties such as seeding-driven formation, amyloid-tropic dye binding, intermolecular β-sheet architecture and cytotoxicity on mammalian cells. During protein deposition, exposed hydrophobic patches force intermolecular clustering and aggregation but these aggregation determinants coexist with properly folded stretches, exhibiting native-like secondary structure. Several reports indicate that inclusion bodies formed by different enzymes or fluorescent proteins show detectable biological activity. By using an engineered green fluorescent protein as reporter we have examined how the cell quality control distributes such active but misfolded protein species between the soluble and insoluble cell fractions and how aggregation determinants act in cells deficient in quality control functions. Most of the tested genetic deficiencies in different cytosolic chaperones and proteases (affecting DnaK, GroEL, GroES, ClpB, ClpP and Lon at different extents) resulted in much less soluble but unexpectedly more fluorescent polypeptides. The enrichment of aggregates with fluorescent species results from a dramatic inhibition of ClpP and Lon-mediated, DnaK-surveyed green fluorescent protein degradation, and it does not perturb the amyloid-like architecture of inclusion bodies. Therefore, the Escherichia coli quality control system promotes protein solubility instead of conformational quality through an overcommitted proteolysis of aggregation-prone polypeptides, irrespective of their global conformational status and biological properties.     

Protein Folding, Misfolding, and Aggregation. Formation of Inclusion Bodies and Aggresomes

In this review the mechanisms of protein folding, misfolding, and aggregation as well as the mechanisms of cell defense against toxic protein aggregates are considered. Misfolded and aggregated proteins in cells are exposed to chaperone-mediated refolding and are degraded by proteasomes if refolding is impossible. Proteolysis-stable protein aggregates accumulate, forming inclusion bodies. In eucaryotic cells, protein aggregates form structures in the pericentrosomal area that have been termed "aggresomes". Formation of aggresomes in cells is a general cellular response to the presence of misfolded proteins when the degrading capacity of the cells is exceeded. The role of aggresomes in disturbance of the proteasomal system operation and in cellular death, particularly in the so-called "protein conformational diseases", is discussed.     

Aggresomes, inclusion bodies and protein aggregation

Intracellular and extracellular accumulation of aggregated protein are linked to many diseases, including ageing-related neurodegeneration and systemic amyloidosis. Cells avoid accumulating potentially toxic aggregates by mechanisms including the suppression of aggregate formation by molecular chaperones and the degradation of misfolded proteins by proteasomes. Once formed, aggregates tend to be refractory to proteolysis and to accumulate in inclusion bodies. This accumulation has been assumed to be a diffusion-limited process, but recent studies suggest that, in animal cells, aggregated proteins are specifically delivered to inclusion bodies by dynein-dependent retrograde transport on microtubules. This microtubule-dependent inclusion body is called an aggresome.     

Decreased proteolysis caused by protein aggregates, inclusion bodies, plaques, lipofuscin, ceroid, and 'aggresomes' during oxidative stress, aging, and disease

Protein aggregation seems to be a common feature of several neurodegenerative diseases and to some extent of physiological aging. It is not always clear why protein aggregation takes place, but a disturbance in the homeostasis between protein synthesis and protein degradation seems to be important. The result is the accumulation of modified proteins, which tend to form high molecular weight aggregates. Such aggregates are also called inclusion bodies, plaques, lipofuscin, ceroid, or ‘aggresomes’ depending on their location and composition. Such aggregates are not inert metabolic end products, but actively influence the metabolism of cells, in particular proteasomal activity and protein turnover. In this review we focus on the influence of oxidative stress on protein turnover, protein aggregate formation and the various interactions of protein aggregates with the proteasome. Furthermore, the formation and effects of protein aggregates during aging and neurodegeneration will be highlighted.     

Fundamental insights in early-stage inclusion body formation

Early-stage inclusion body formation is still mysterious. Literature is ambiguous about the existence of rod-shaped protein aggregates, a potential sponge-like inclusion body scaffold as well as the number of inclusion bodies per Escherichia coli cell. In this study, we verified the existence of rod-shaped inclusion bodies, confirmed their porous morphology, the presence of multiple protein aggregates per cell and modelled inclusion body formation as function of the number of generations.     

Localization of Functional Polypeptides in Bacterial Inclusion Bodies

Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a β-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular β-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.     

Routes to active proteins from transformed microorganisms.

Over-expression of recombinant proteins in microbial hosts results in the formation of active soluble protein or of insoluble aggregates (inclusion bodies). Efficient in vitro refolding strategies have been developed to reactivate inactive proteins from inclusion bodies. Co-expression of molecular chaperones may provide a tool to promote correct structure formation of recombinant proteins in vivo.     

Dark proteins: effect of inclusion body formation on quantification of protein expression

Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used calibrated fluorescent intensity measurements to determine the average number of active EGFP present per cell. Both measurements were carried out as a function of cellular doubling time, over a range of 45-75 min. We found that the ratio of inclusion body EGFP to active EGFP varied strongly as a function of the cellular growth rate, and that the number of "dark" proteins in the aggregates could in fact be substantial, reaching ratios as high as approximately five proteins locked into inclusion bodies for every active protein (at the fastest growth rate), and dropping to ratios well below 1 (for the slowest growth rate). Our results suggest that efforts to compare computational models to protein numbers derived from fluorescence measurements should take inclusion body loss into account, especially when working with rapidly growing cells.     

Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in inclusion bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within inclusion bodies. We describe here the use of Fourier transform infrared spectroscopy for the structural analysis of inclusion bodies both purified from cells and in vivo. Examples are reported concerning the study of kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co-expression of chaperones.     

Localization of functional polypeptides in bacterial inclusion bodies

Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a beta-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular beta-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.     

The chaperone DnaK controls the fractioning of functional protein between soluble and insoluble cell fractions in inclusion body-forming cells

Background  

The molecular mechanics of inclusion body formation is still far from being completely understood, specially regarding the occurrence of properly folded, protein species that exhibit natural biological activities. We have here comparatively explored thermally promoted, in vivo protein aggregation and the formation of bacterial inclusion bodies, from both structural and functional sides. Also, the status of the soluble and insoluble protein versions in both aggregation systems have been examined as well as the role of the main molecular chaperones GroEL and DnaK in the conformational quality of the target polypeptide.     

Towards revealing the structure of bacterial inclusion bodies

Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6–12 nm, they are comprised of residue-specific cross-β structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies.Key words: bacterial, inclusion bodies, amyloid fibrils, protein aggregation, amyloid-like, nuclear magnetic resonance, electron microscope, X-ray diffraction, hydrogen/deuterium exchange, cross-β     

Aggresomes do not represent a general cellular response to protein misfolding in mammalian cells

Background

Aggresomes are juxtanuclear inclusion bodies that have been proposed to represent a general cellular response to misfolded proteins in mammalian cells. Yet, why aggresomes are not a pathological characteristic of protein misfolding diseases is unclear. Here, we investigate if a misfolded protein inevitably forms aggresomes in mammalian cells.

Results

We show that a cytoplasmic form of the prion protein may form aggresomes or dispersed aggregates in different cell lines. In contrast to aggresomes, the formation of dispersed aggregates is insensitive to histone deacetylase 6 inhibitors and does not result in cytoskeleton rearrangements. Modulation of expression levels or proteasome inhibitors does not alter the formation of dispersed aggregates.

Conclusion

Our results establish that aggresomes are not obligatory products of protein misfolding in vivo.     

Expression in Escherichia coli and in vitro refolding of the plant transcription factor Arabidopsis thaliana RGL3

Recombinant Arabidopsis thaliana (At) RGL-3, using two vectors pMAL-c2 and pET 21, was expressed as inclusion bodies in Escherichia coli under a range of temperature conditions. Only low levels (8-12% of total protein) of soluble protein were produced. The "soluble" fraction was shown by native PAGE to exist as soluble aggregates of RGL-3. A method was developed, consisting of induction of expression at various temperatures that yielded high levels of refoldable inclusion bodies using the pET vector. (At) RGL-3, as inclusion bodies, was solubilized in 8M urea and refolding was initiated by 20-fold direct dilution of denaturant. Under optimal conditions, 87% of the denatured protein of inclusion bodies was successfully re-natured. Refolding was monitored by "native" PAGE. Refolded RGL-3 was shown to be present as monomers and dimers. Attempts to further purify His-tagged RGL-3 using Ni/NTA chromatography resulted in the formation of higher polymers.     

Cytotoxic Aggregation and Amyloid Formation by the Myostatin Precursor Protein

Myostatin, a negative regulator of muscle growth, has been implicated in sporadic inclusion body myositis (sIBM). sIBM is the most common age-related muscle-wastage disease with a pathogenesis similar to that of amyloid disorders such as Alzheimer''s and Parkinson''s diseases. Myostatin precursor protein (MstnPP) has been shown to associate with large molecular weight filamentous inclusions containing the Alzheimer''s amyloid beta peptide in sIBM tissue, and MstnPP is upregulated following ER stress. The mechanism for how MstnPP contributes to disease pathogenesis is unknown. Here, we show for the first time that MstnPP is capable of forming amyloid fibrils in vitro. When MstnPP-containing Escherichia coli inclusion bodies are refolded and purified, a proportion of MstnPP spontaneously misfolds into amyloid-like aggregates as characterised by electron microscopy and binding of the amyloid-specific dye thioflavin T. When subjected to a slightly acidic pH and elevated temperature, the aggregates form straight and unbranched amyloid fibrils 15 nm in diameter and also exhibit higher order amyloid structures. Circular dichroism spectroscopy reveals that the amyloid fibrils are dominated by β-sheet and that their formation occurs via a conformational change that occurs at a physiologically relevant temperature. Importantly, MstnPP aggregates and protofibrils have a negative effect on the viability of myoblasts. These novel results show that the myostatin precursor protein is capable of forming amyloid structures in vitro with implications for a role in sIBM pathogenesis.     

Bacterial inclusion bodies are cytotoxic in vivo in absence of functional chaperones DnaK or GroEL

Cytotoxicity of cytoplasmic bacterial inclusion bodies has been explored in vivo in cells producing a model, misfolding-prone beta-galactosidase fusion protein. The formation of such aggregates does not result in detectable toxicity on Escherichia coli producing cells. However, a deficiency in the main chaperones DnaK or GroEL but not in other components of the heat shock system such as the chaperone ClpA or the protease Lon, promotes a dramatic inhibition of cell growth. The role of DnaK and GroEL in minimizing toxicity of in vivo protein aggregation is discussed in the context of the conformational stress and the protein quality control system.     

Construction and deconstruction of bacterial inclusion bodies

Bacterial inclusion bodies (IBs) are refractile aggregates of protease-resistant misfolded protein that often occur in recombinant bacteria upon gratuitous overexpression of cloned genes. In biotechnology, the formation of IBs represents a main obstacle for protein production since even favouring high protein yields, the in vitro recovery of functional protein from insoluble deposits depends on technically diverse and often complex re-folding procedures. On the other hand, IBs represent an exciting model to approach the in vivo analysis of protein folding and to explore aggregation dynamics. Recent findings on the molecular organisation of embodied polypeptides and on the kinetics of inclusion body formation have revealed an unexpected dynamism of these protein aggregates, from which polypeptides are steadily released in living cells to be further refolded or degraded. The close connection between in vivo protein folding, aggregation, solubilisation and proteolytic digestion offers an integrated view of the bacterial protein quality control system of which IBs might be an important component especially in recombinant bacteria.     

Mallory-Denk-bodies: lessons from keratin-containing hepatic inclusion bodies

Inclusion bodies are characteristic morphological features of various neuronal, muscular and other human disorders. They share common molecular constituents such as p62, chaperones and proteasome subunits. The proteins within aggregates are misfolded with increased beta-sheet structure, they are heavily phosphorylated, ubiquitinylated and partially degraded. Furthermore, involvement of proteasomal system represents a common feature of virtually all inclusions. Multiple aggregates contain intermediate filament proteins as their major constituents. Among them, Mallory-Denk bodies (MDBs) are the best studied. MDBs represent hepatic inclusions observed in diverse chronic liver diseases such as alcoholic and non-alcoholic steatohepatitis, chronic cholestasis, metabolic disorders and hepatocellular neoplasms. MDBs are induced in mice fed griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine and resolve after discontinuation of toxin administration. The availability of a drug-induced model makes MDBs a unique tool for studying inclusion formation. Our review summarizes the recent advances gained from this model and shows how they relate to observations in other aggregates. The MDB formation-underlying mechanisms include protein misfolding, chaperone alterations, disproportional protein expression with keratin 8>keratin 18 levels and subsequent keratin 8 crosslinking via transglutaminase. p62 presence is crucial for MDB formation. Proteasome inhibitors precipitate MDB formation, whereas stimulation of autophagy with rapamycin attenuates their formation.     

Ubiquitin versus misfolding: The minimal requirements for inclusion body formation

Ubiquitin-containing inclusion bodies are characteristic features of numerous neurodegenerative diseases, but whether ubiquitin plays a functional role in the formation of these protein deposits is unclear. In this issue, Bersuker et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201511024) report that protein misfolding without ubiquitylation is sufficient for translocation into inclusion bodies.A large number of sporadic and familial neurodegenerative diseases that differ in their age of onset and manifestation share striking pathological features at the cellular level, suggesting that a common etiology may be responsible for the demise of neurons. Most notable is the aggregation of improperly folded proteins in affected neurons in these so-called protein misfolding diseases that include Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob disease, as well as amyotrophic lateral sclerosis and other motor neuron diseases. Protein aggregates are inherently toxic for cells, underscoring their candidate status as a common denominator in these diseases (Bucciantini et al., 2002). A causative role for aberrant protein conformations is further strengthened by the existence of a family of rare, inheritable neurodegenerative disorders, which are a direct consequence of expansions of polyglutamine repeats that render the mutant proteins prone to aggregation. Given that neuronal cells often must last an organism’s lifetime with little opportunity to dilute protein waste through cell division, it is not hard to imagine that they are particularly susceptible to the gradual accumulation of aberrant proteins that favor precipitation in insoluble protein aggregates.Neurons and other cells have three major lines of defense to minimize the damage that aggregation-prone proteins can cause to cellular homeostasis (Fig. 1). The first two are based on a seek-and-destroy strategy in which the two main intracellular proteolytic systems play complementary roles. Although monomeric aberrant proteins are efficiently targeted for hydrolysis in proteasomes, these proteolytic complexes are unable to process oligomeric protein aggregates (Verhoef et al., 2002). Destruction of proteins in proteasomes requires complete unfolding of the deemed proteins, which may be hard, if not impossible, in the case of tightly associated misfolded proteins. Macroautophagy, however, is a proteolytic pathway that is able to process oligomeric misfolded proteins, as it involves the capturing of cytosolic constituents, including macromolecular complexes like protein aggregates, in double-membrane vesicles that fuse with lysosomes (Ravikumar et al., 2004). As such, macroautophagy complements proteasomal degradation in keeping the cellular environment free from toxic protein species.Open in a separate windowFigure 1.Three lines of defense against misfolded proteins. There are three protective mechanisms that are involved in minimizing the toxicity of misfolded proteins: proteasomal degradation (I), macroautophagic clearance (II), and inclusion body formation (III). Ubiquitin is linked to each of these processes, as it can target proteins for proteasomal and macroautophagosomal degradation and is enriched in inclusion bodies.In the unfortunate case that the production of aggregation-prone proteins exceeds the capacity of both the proteasomal and lysosomal systems, a potential catastrophic situation arises as misfolded proteins may precipitate in large, insoluble aggregates. In these cases, a third protective mechanism can come to the rescue and primarily provides damage control, as it intercepts protein aggregates and sequesters them in dedicated subcellular structures, thereby minimizing the harm that the aberrant proteins may cause (Johnston et al., 1998). It is this process that is responsible for the formation of the characteristic inclusion bodies that are typically observed in affected neurons and known under different names depending on the neurodegenerative disorder in which they occur, such as Lewy bodies in Parkinson’s disease, Bunina bodies in amyotrophic lateral sclerosis, and intranuclear inclusions in several polyglutamine disorders (Alves-Rodrigues et al., 1998). Although these structures were originally considered as a potential cause for the cellular pathology, a large body of evidence suggests that they actually lessen the cellular damage caused by toxic proteins (Arrasate et al., 2004). Although their presence may not be without negative consequence for the cells, the controlled formation of waste deposits may be the best possible option for the cell when facing excessive amounts of aggregated proteins.Interestingly, the protein modifier ubiquitin, a posttranslational modification covalently linked to lysine residues of target proteins, appears to be somehow involved in each of these three protective mechanisms. Polyubiquitylation, or conjugation of a chain of ubiquitin molecules, targets proteins for proteasomal degradation and is likewise also critical for proteasomal destruction of misfolded proteins (Kleiger and Mayor, 2014). Even though macroautophagy was originally seen as a nonselective catabolic pathway, more recent studies have suggested that it also involves a high level of specificity with ubiquitin chains being an important substrate recruitment signal (Kraft et al., 2010). In sharp contrast to the well-defined targeting function of ubiquitin in these proteolytic mechanisms, its possible role in the formation of inclusion bodies has been less clear. This is somewhat ironic, given that the initial observations of ubiquitin-positive inclusions in neurodegeneration date back almost three decades (Mori et al., 1987) and have been among the main findings that sparked the interest in a possible role of dysfunctional ubiquitin-dependent proteasomal degradation in neurodegenerative disorders (Cummings et al., 1998). In this issue, Bersuker et al. revisited this important question using an elegant system that allowed them to follow specifically designed reporter proteins that could be switched from folded to misfolded states by administration of cell-permeable ligands. Using this approach, they confirmed that introducing a misfolded state resulted in rapid clearance of the reporter proteins by ubiquitin-dependent proteasomal degradation, the first line of defense against misfolded proteins. Consistent with the prevailing model, they also found that the misfolded reporters accumulated in inclusion bodies when they increased the load of aggregation-prone proteins by simultaneously expressing a fragment of mutant huntingtin containing an expanded polyglutamine repeat, the protein responsible for Huntington’s disease. Interestingly, chemical inhibition of the ubiquitin activase, an enzyme that is critical for ubiquitin conjugation, showed that translocation of the reporter proteins to inclusion bodies did not require ubiquitylation, arguing that the misfolded state is sufficient to reach the final destination.If ubiquitin is not needed for targeting misfolded proteins to inclusion bodies, why then do these proteinaceous deposits contain such large amounts of ubiquitin? The fact that ubiquitin is not required for the recruitment of misfolded proteins to inclusion bodies does not exclude the possibility that ubiquitylation targets properly folded proteins to inclusion bodies. Thus, a possible scenario is that inclusion bodies, once they have been seeded by the ubiquitin-independent sequestration of misfolded proteins, will start to gather soluble polyubiquitylated proteins that typically accumulate under conditions of disturbed protein homeostasis. The authors investigated this possibility by expressing a reporter substrate that contained a degradation signal and was therefore efficiently targeted for ubiquitin-dependent proteasomal degradation. Interestingly, even though these substrates accumulated in a ubiquitylated form when proteasomal degradation was obstructed, they did not localize to the inclusion bodies that otherwise gathered misfolded reporters. This suggests that ubiquitin chains—at least those that target substrates for proteasomal degradation—are not sufficient to autonomously target proteins to inclusion bodies and, at the same time, excludes the possibility that their presence is due to a general sequestration of ubiquitylated proteasome substrates.Alternatively, ubiquitin in inclusions may reflect an attempt of the cell to get rid of the sequestrated protein aggregates once they have reached the inclusion body by targeting them for destruction via ubiquitin-dependent proteolytic systems. Indeed, in vivo studies suggest that inclusion bodies are not a dead-end product but can be cleared from affected neurons (Yamamoto et al., 2000). Even though ubiquitin-dependent autophagosomal and proteasomal degradation are primary candidates for facilitating disposal of inclusions (Martín-Aparicio et al., 2001; Wong et al., 2008), it should be noted that it is presently unclear how this would be mechanistically executed. The data presented by Bersuker et al. (2016) show that the pool of ubiquitin in inclusion bodies is rather static, arguing against a direct role in the turnover, if any, of the ubiquitylated proteins present in the inclusions.Where do these findings leave us? It is fair to say that the functional significance of ubiquitin in inclusion bodies remains somewhat elusive. Following the road of exclusion as in the present study, we can put a solid strike through several trivial explanations for the presence of ubiquitin in inclusions, but further research will be needed to get a more definitive answer about ubiquitin’s role in this process or the lack thereof. It also brings up questions about the role of the microtubule-associated deacetylase HDAC6 in this process. Some studies have provided data that support an essential role for this cytosolic deacetylase in transporting aggregates to inclusions by virtue of its ability to simultaneously bind ubiquitin conjugates and the dynein motors that are required for their sequestration (Kawaguchi et al., 2003; Olzmann et al., 2007). However, HDAC6 has also been linked to degradation of aggregation-prone proteins by macroautophagy, suggesting that it may indirectly influence the kinetics of inclusion body formation (Pandey et al., 2007; Lee et al., 2010). Even though these processes are not mutually exclusive and may well be functionally linked, the present findings motivate a closer look at the molecular mechanisms that link HDAC6 to the formation of inclusion bodies. It should be noted that although the presented data demonstrate that the canonical ubiquitin chains that target proteins for proteasomal degradation are insufficient to promote their translocation to inclusion bodies, it does not exclude implication of alternative ubiquitin chains. Ubiquitin modifications come in many different flavors, and, in particular, the K63-linked polyubiquitin chains, which do not target for proteasomal degradation, have been linked to both macroautophagy and inclusion body formation (Lim and Lim, 2011).The present work also underscores the importance of the exclusive role of protein aggregation in directing misfolded proteins to inclusion bodies. This finding resonates with an earlier study from the same group, in which they reported that targeting of misfolded proteins for autophagy is a direct consequence of their aggregation and does not necessarily require ubiquitylation (Riley et al., 2010). A picture starts to emerge of a general strategy in which the attention of these protective mechanisms is directly drawn to the problematic proteins by the very same virtue that causes their misbehavior, namely their tendency to aggregate. The central role of protein aggregation, as opposed to ubiquitylation, may also be relevant for the similarities and dissimilarities between the formation of inclusion bodies in the cytosolic and nuclear compartments of cells. Whereas the present study probes into the role of ubiquitin in the generation of cytosolic inclusions, intranuclear inclusions are most notoriously associated with the pathology of neurodegenerative diseases. Even though there are fundamental differences in ubiquitin targeting and transport mechanisms between these compartments, the intrinsic property of the proteins to aggregate applies to both, and it is also feasible that in the nucleus, the misfolded domains suffice to facilitate their translocation to inclusion bodies. The lack of a need for a middleman in this critical process may reflect the archaic nature of this innate response and allow rapid incapacitation of these inherently toxic species. This will also ensure that handling of these proteins is not susceptible to disturbed ubiquitin homeostasis, as often is the case in neurodegenerative disorders.     

Role of molecular chaperones in inclusion body formation

Protein misfolding and aggregation are linked to several degenerative diseases and are responsible for the formation of bacterial inclusion bodies. Roles of molecular chaperones in promoting protein deposition have been speculated but not proven in vivo. We have investigated the involvement of individual chaperones in inclusion body formation by producing the misfolding-prone but partially soluble VP1LAC protein in chaperone null bacterial strains. Unexpectedly, the absence of a functional GroEL significantly reduced aggregation and favoured the incidence of the soluble protein form, from 4 to 35% of the total VP1LAC protein. On the other hand, no regular inclusion bodies were then formed but more abundant small aggregates up to 0.05 microm(3). Contrarily, in a DnaK(-) background, the amount of inclusion body protein was 2.5-fold higher than in the wild-type strain and the average volume of the inclusion bodies increased from 0.25 to 0.38 microm(3). Also in the absence of DnaK, the minor fraction of soluble protein appears as highly proteolytically stable, suggesting an inverse connection between proteolysis and aggregation managed by this chaperone. In summary, GroEL and DnaK appear as major antagonist controllers of inclusion body formation by promoting and preventing, respectively, the aggregation of misfolded polypeptides. GroEL might have, in addition, a key role in driving the protein transit from the soluble to the insoluble cell fraction and also in the opposite direction. Although chaperones ClpB, ClpA, IbpA and IbpB also participate in these processes, the impact of the respective null mutations on bacterial inclusion body formation is much more moderate.