Bacterial inclusion bodies contain amyloid-like structure.
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ABSTRACT:
SUBMITTER: Wang L
PROVIDER: S-EPMC2494559 | biostudies-literature | 2008 Aug
REPOSITORIES: biostudies-literature
Publications
Bacterial inclusion bodies contain amyloid-like structure.
Wang Lei L Maji Samir K SK Sawaya Michael R MR Eisenberg David D Riek Roland R
PLoS biology 20080801 8
Protein aggregation is a process in which identical proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as amorphous, lacking any long-range order, or highly ordered fibrils. Protein fibrils can be composed of native globular molecules, such as the hemoglobin molecules in sickle-cell fibrils, or can be reorganized beta-sheet-rich aggregates, termed amyloid-like fibrils. Amyloid fibrils are associated with several pathological conditions ...[more]
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Project description:The formation of amyloid aggregates is related to the onset of a number of human diseases. Recent studies provide compelling evidence for the existence of related fibrillar structures in bacterial inclusion bodies (IBs). Bacteria might thus provide a biologically relevant and tuneable system to study amyloid aggregation and how to interfere with it. Particularly suited for such studies are protein models for which structural information is available in both IBs and amyloid states. The only high-resolution structure of an infectious amyloid state reported to date is that of the HET-s prion forming domain (PFD). Importantly, recent solid-state NMR data indicates that the structure of HET-s PFD in IBs closely resembles that of the infectious fibrils. Here we present an exhaustive conformational characterization of HET-s IBs in order to establish the aggregation of this prion in bacteria as a consistent cellular model in which the effect of autologous or heterologous protein quality machineries and/or anti-aggregational and anti-prionic drugs can be further studied.
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Project description:BACKGROUND: Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. RESULTS: Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. CONCLUSIONS: An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity.
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Project description:Bacterial inclusion bodies (IBs) are discrete macromolecular complexes that appear in recombinant prokaryotic cells under stress conditions. These structures are often discarded for biotechnological uses given the difficulty in recovering proteins of interest from them in a soluble form. However, recent approaches have revealed the potential of these protein clusters as biomaterials to promote cell growth and as protein depots for the release of recombinant proteins for biotechnological and biomedical applications. Although these kinds of natural supramolecular complexes have attracted great interest, no comprehensive study of their toxicity in cell cultures has been carried out. In this study, caco-2 cells were exposed to natural IBs, soluble protein-only nanoparticles (NPs), and non-assembled versions of the same protein for comparative purposes. Cytotoxicity, oxidative stress, and genotoxicity were analyzed for all these protein formats. Natural IBs and soluble protein formats demonstrated their safety in eukaryotic cells. No cytotoxicity, genotoxicity, or oxidative stress was detected in caco-2 cells exposed to the protein samples in any of the experimental conditions evaluated, which covered protein concentrations used in previous biological activity assays. These conditions evaluated the activity of protein samples obtained from three prokaryotic hosts [Escherichia coli and the endotoxin-free expression systems Lactococcus lactis and ClearColi® BL21 (DE3)]. Our results demonstrate that natural IBs and soluble protein nanoparticles are non-toxic materials for eukaryotic cells and that this may represent an interesting alternative to the classical unassembled format of recombinant proteins for certain applications in biotechnology and biomedicine.
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Project description:High level expression of recombinant proteins in bacteria often results in their aggregation into inclusion bodies. Formation of inclusion bodies poses a major bottleneck in high-throughput recovery of recombinant protein. These aggregates have amyloid-like nature and can retain biological activity. Here, effect of expression temperature on the quality of Escherichia coli asparaginase II (a tetrameric protein) inclusion bodies was evaluated. Asparaginase was expressed as inclusion bodies at different temperatures. Purified inclusion bodies were checked for biological activities and analyzed for structural properties in order to establish a structure-activity relationship. Presence of activity in inclusion bodies showed the existence of properly folded asparaginase tetramers. Expression temperature affected the properties of asparaginase inclusion bodies. Inclusion bodies expressed at higher temperatures were characterized by higher biological activity and less amyloid content as evident by Thioflavin T binding and Fourier Transform Infrared (FTIR) spectroscopy. Complex kinetics of proteinase K digestion of asparaginase inclusion bodies expressed at higher temperatures indicate higher extent of conformational heterogeneity in these aggregates.
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Project description:Experiments were performed to test the hypothesis that recombinant human uncoupling protein-2 (UCP2) ectopically expressed in bacterial inclusion bodies binds nucleotides in a manner identical with the nucleotide-inhibited uncoupling that is observed in kidney mitochondria. For this, sarkosyl-solubilized UCP2 inclusion bodies were treated with the polyoxyethylene ether detergent C12E9 and hydroxyapatite. Protein recovered from hydroxyapatite chromatography was approx. 90% pure UCP2, as judged by Coomassie Blue and silver staining of polyacrylamide gels. Using fluorescence resonance energy transfer, N-methylanthraniloyl-tagged purine nucleoside di- and tri-phosphates exhibited enhanced fluorescence with purified UCP2. Dissociation constants determined by least-squares non-linear regression indicated that the affinity of UCP2 for these fluorescently tagged nucleotides was 3-5 microM or perhaps an order of magnitude stronger, depending on the model used. Competition experiments with [8-14C]ATP demonstrated that UCP2 binds unmodified purine and pyrimidine nucleoside triphosphates with 2-5 microM affinity. Affinities for ADP and GDP were approx. 10-fold lower. These data indicate that: UCP2 (a) is at least partially refolded from sarkosyl-solubilized bacterial inclusion bodies by a two-step treatment with C12E9 detergent and hydroxyapatite; (b) binds purine and pyrimidine nucleoside triphosphates with low micromolar affinity; (c) binds GDP with the same affinity as GDP inhibits superoxide-stimulated uncoupling by kidney mitochondria; and (d) exhibits a different nucleotide preference than kidney mitochondria.
| S-EPMC1222799 | biostudies-other
Project description:The formation of spore-filled fruiting bodies by myxobacteria is a fascinating case of multicellular self-organization by bacteria. The organization of Myxococcus xanthus into fruiting bodies has long been studied not only as an important example of collective motion of bacteria, but also as a simplified model for developmental morphogenesis. Sporulation within the nascent fruiting body requires signaling between moving cells in order that the rod-shaped self-propelled cells differentiate into spores at the appropriate time. Probing the three-dimensional structure of myxobacteria fruiting bodies has previously presented a challenge due to limitations of different imaging methods. A new technique using Infrared Optical Coherence Tomography (OCT) revealed previously unknown details of the internal structure of M. xanthus fruiting bodies consisting of interconnected pockets of relative high and low spore density regions. To make sense of the experimentally observed structure, modeling and computer simulations were used to test a hypothesized mechanism that could produce high-density pockets of spores. The mechanism consists of self-propelled cells aligning with each other and signaling by end-to-end contact to coordinate the process of differentiation resulting in a pattern of clusters observed in the experiment. The integration of novel OCT experimental techniques with computational simulations can provide new insight into the mechanisms that can give rise to the pattern formation seen in other biological systems such as dictyostelids, social amoeba known to form multicellular aggregates observed as slugs under starvation conditions.
| S-EPMC3531287 | biostudies-literature
Project description:The acylphosphatase from Sulfolobus solfataricus (Sso AcP) is a globular protein able to aggregate in vitro from a native-like conformational ensemble without the need for a transition across the major unfolding energy barrier. This process leads to the formation of assemblies in which the protein retains its native-like structure, which subsequently convert into amyloid-like aggregates. Here, we investigate the mechanism by which Sso AcP aggregates in vivo to form bacterial inclusion bodies after expression in E. coli. Shortly after the initiation of expression, Sso AcP is incorporated into inclusion bodies as a native-like protein, still exhibiting small but significant enzymatic activity. Additional experiments revealed that this overall process of aggregation is enhanced by the presence of the unfolded N-terminal region of the sequence and by destabilization of the globular segment of the protein. At later times, the Sso AcP molecules in the inclusion bodies lose their native-like properties and convert into β-sheet-rich amyloid-like structures, as indicated by their ability to bind thioflavin T and Congo red. These results show that the aggregation behavior of this protein is similar in vivo to that observed in vitro, and that, at least for a predominant part of the protein population, the transition from a native to an amyloid-like structure occurs within the aggregate state.
| S-EPMC5479110 | biostudies-literature
Project description:BackgroundThe formation of protein inclusions is connected to the onset of many human diseases. Human RNA binding proteins containing intrinsically disordered regions with an amino acid composition resembling those of yeast prion domains, like TDP-43 or FUS, are being found to aggregate in different neurodegenerative disorders. The structure of the intracellular inclusions formed by these proteins is still unclear and whether these deposits have an amyloid nature or not is a matter of debate. Recently, the aggregation of TDP-43 has been modelled in bacteria, showing that TDP-43 inclusion bodies (IBs) are amorphous but intrinsically neurotoxic. This observation raises the question of whether it is indeed the lack of an ordered structure in these human prion-like protein aggregates the underlying cause of their toxicity in different pathological states.ResultsHere we characterize the IBs formed by the human prion-like RNA-processing protein HNRPDL. HNRPDL is linked to the development of limb-girdle muscular dystrophy 1G and shares domain architecture with TDP-43. We show that HNRPDL IBs display characteristic amyloid hallmarks, since these aggregates bind to amyloid dyes in vitro and inside the cell, they are enriched in intermolecular β-sheet conformation and contain inner amyloid-like fibrillar structure. In addition, despite their ordered structure, HNRPDL IBs are highly neurotoxic.ConclusionsOur results suggest that at least some of the disorders caused by the aggregation of human prion-like proteins would rely on the formation of classical amyloid assemblies rather than being caused by amorphous aggregates. They also illustrate the power of microbial cell factories to model amyloid aggregation.
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Project description:BackgroundRecombinant protein expression in bacteria often leads to the formation of intracellular insoluble protein deposits, a major bottleneck for the production of soluble and active products. However, in recent years, these bacterial protein aggregates, commonly known as inclusion bodies (IBs), have been shown to be a source of stable and active protein for biotechnological and biomedical applications. The formation of these functional IBs is usually facilitated by the fusion of aggregation-prone peptides or proteins to the protein of interest, leading to the formation of amyloid-like nanostructures, where the functional protein is embedded.ResultsIn order to offer an alternative to the classical amyloid-like IBs, here we develop functional IBs exploiting the coiled-coil fold. An in silico analysis of coiled-coil and aggregation propensities, net charge, and hydropathicity of different potential tags identified the natural homo-dimeric and anti-parallel coiled-coil ZapB bacterial protein as an optimal candidate to form assemblies in which the native state of the fused protein is preserved. The protein itself forms supramolecular fibrillar networks exhibiting only α-helix secondary structure. This non-amyloid self-assembly propensity allows generating innocuous IBs in which the recombinant protein of interest remains folded and functional, as demonstrated using two different fluorescent proteins.ConclusionsHere, we present a proof of concept for the use of a natural coiled-coil domain as a versatile tool for the production of functional IBs in bacteria. This α-helix-based strategy excludes any potential toxicity drawback that might arise from the amyloid nature of β-sheet-based IBs and renders highly active and homogeneous submicrometric particles.
| S-EPMC7268670 | biostudies-literature
Project description:Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.
| S-EPMC3907574 | biostudies-literature