Figure 3 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Effect of lysozyme concentration on the protein-ribosome coaggregation process. (A) Time course of change in turbidity when lysozyme at two different protein concentrations was incubated with 20 mM DTT in absence and presence of 70S ribosome: 10 μ M lysozyme + 20 mM DTT (◾ ), 65 μ M lysozyme + 20 mM DTT ( ), 10 μ M lysozyme + 20 mM DTT + 0.1 μ M 70S ( ), 65 μ M lysozyme + 20 mM DTT + 0.65 μ M 70S ( ). Lysozyme reduced with 20 mM DTT has been abbreviated as Lyso (R). (B)i) 2 μ M, 10 μ M or 65 μ M of lysozyme was incubated in 20 mM DTT in the presence of 0.1 μ M 70S ribosome for 45 minutes at room temperature, centrifuged and the resuspended pellets (in 40 μ l of Buffer A) were loaded on a 12% SDS PAGE. Lysozyme reduced with 20 mM DTT has been abbreviated as Lyso (R). ii) 2 μ M, 10 μ M or 65 μ M of lysozyme was incubated in 20 mM DTT in the presence of 0.1 μ M 70S ribosome for 45 minutes at room temperature, pelleted down and the resuspended pellets (in 25 μ l Buffer A containing 1 M urea) were loaded on a 1% agarose gel. Lanes from left to right for both the gels contain: 2 μ M lysozyme + 20 mM DTT + 70S, 10 μ M lysozyme + 20 mM DTT + 70S, 65 μ M lysozyme + 20 mM DTT + 70S. (C) 65 μ M of lysozyme was incubated in 20 mM DTT for 30 min, centrifuged and the pellet obtained was incubated in Buffer A with or without 0.65 μ M 70S in the presence of DTT for 45 minutes at room temperature. After incubation samples were centrifuged, aggregate resuspended in 40 μ l of Buffer A and loaded on a 12% SDS-PAGE. Left to right lane: Lyso (T): 65 μ M lysozyme (Total protein), Lyso (A): aggregate of 65 μ M of lysozyme incubated with 20 mM DTT for 30 min (pellet), 70S + Lyso (A): Lyso (A) + 20 mM DTT + 0.65 μ M 70S (pellet).
Source publication
An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subu...
Contexts in source publication
Context 1
... It is well estab- lished that an increase in protein concentration leads to increased protein aggregation. Hence, further studies were conducted to observe whether the facilitation of protein-protein interaction at higher protein concentration could lead to reduced lysozyme-ribosome interaction and hence reduced ribosome aggregation. As shown in Fig. 3A, a comparable increase in turbidity was observed when lysozyme at a concentration of 65 μ M was incu- bated in 20 mM DTT either in the absence or in the presence of the ribosome (0.65 μ M) (Fig. 3A).This initially suggested that the ribosome aggregation was circumvented at high protein concentrations. However, SDS-PAGE analysis still ...
Context 2
... interaction at higher protein concentration could lead to reduced lysozyme-ribosome interaction and hence reduced ribosome aggregation. As shown in Fig. 3A, a comparable increase in turbidity was observed when lysozyme at a concentration of 65 μ M was incu- bated in 20 mM DTT either in the absence or in the presence of the ribosome (0.65 μ M) (Fig. 3A).This initially suggested that the ribosome aggregation was circumvented at high protein concentrations. However, SDS-PAGE analysis still revealed the presence of ribosomal components in the aggregate formed when 2 μ M, 10 μ M or 65 μ M of lysozyme (with DTT) was incubated with 0.1 μ M of 70S ribosome. This observation suggested that ...
Context 3
... analysis still revealed the presence of ribosomal components in the aggregate formed when 2 μ M, 10 μ M or 65 μ M of lysozyme (with DTT) was incubated with 0.1 μ M of 70S ribosome. This observation suggested that turbidity measurements at 450 nm were unable to register the lysozyme induced ribosome aggregation at higher protein concentrations (Fig. 3Bi). Also, the relative intensity of the rRNA in the aggregate obtained upon incubation of 2 μ M, 10 μ M or 65 μ M lysozyme with equivalent amount of ribosome (0.1 μ M), showed increasing rRNA aggre- gation with increasing protein concentrations (Fig. 3Bii). This is possible either if ribosome-protein interaction leading to ribosomal ...
Context 4
... unable to register the lysozyme induced ribosome aggregation at higher protein concentrations (Fig. 3Bi). Also, the relative intensity of the rRNA in the aggregate obtained upon incubation of 2 μ M, 10 μ M or 65 μ M lysozyme with equivalent amount of ribosome (0.1 μ M), showed increasing rRNA aggre- gation with increasing protein concentrations (Fig. 3Bii). This is possible either if ribosome-protein interaction leading to ribosomal aggregation supersedes increased protein-protein interaction or if the aggregate formed is also capable of inducing ribosome trapping and hence its ...
Context 5
... μ M lysozyme was incubated in 20 mM DTT for 30 min. Lysozyme undergoes almost complete aggregation under these conditions. The lysozyme aggregates obtained by centrifugation was incubated with 0.65 μ M ribo- some for 45 minutes. SDS PAGE analysis shows that the preformed lysozyme aggregates also had the ability to stimulate ribosome aggregation (Fig. 3C). The loose and flexible structure of lysozyme aggregates formed under reducing conditions 23 could have enabled protein-ribosome interaction to occur and thereby induced ribosome aggregation. This ability of lysozyme aggregates might contribute significantly to the increased ribosome aggre- gation observed at high protein ...
Context 6
... (Fig. 3C). The loose and flexible structure of lysozyme aggregates formed under reducing conditions 23 could have enabled protein-ribosome interaction to occur and thereby induced ribosome aggregation. This ability of lysozyme aggregates might contribute significantly to the increased ribosome aggre- gation observed at high protein concentration (Fig. 3B) or on a longer time scale at low protein concentration as shown in Fig. 2A. A recent study has also demonstrated that the addition of pathological tau oligomers in an in vitro translation assay leads to significant reduction in translation of the GFP reporter gene and this observa- tion has been attributed to a dysfunctional ...
Similar publications
During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptid...
Citations
... (3) Our data do not fully exclude the possibility of a direct interaction with an element of the ribosome or ribosome-rescue pathway, which could lead to a loss of ribosomal quality control and, eventually, a loss of proteostasis. Although the in vitro translation assays argue against this scenario, it has been shown that ribosomes are prone to coaggregation when confronted with aggregating proteins 42 . The authors of that study proposed that this may be facilitated by charge interactions between the negatively charged ribosomal RNA and the positively charged protein they were studying (lysozyme) 42 . ...
... Although the in vitro translation assays argue against this scenario, it has been shown that ribosomes are prone to coaggregation when confronted with aggregating proteins 42 . The authors of that study proposed that this may be facilitated by charge interactions between the negatively charged ribosomal RNA and the positively charged protein they were studying (lysozyme) 42 . However, despite our P33 peptide being highly charged, the interaction does not take place in the presence of erythromycin, a ribosome inhibitor, suggesting that this is not the mode of action of our peptide. ...
Drug-resistant bacteria pose an urgent global health threat, necessitating the development of antibacterial compounds with novel modes of action. Protein biosynthesis accounts for up to half of the energy expenditure of bacterial cells, and consequently inhibiting the efficiency or fidelity of the bacterial ribosome is a major target of existing antibiotics. Here, we describe an alternative mode of action that affects the same process: allowing translation to proceed but causing co-translational aggregation of the nascent peptidic chain. We show that treatment with an aggregation-prone peptide induces formation of polar inclusion bodies and activates the SsrA ribosome rescue pathway in bacteria. The inclusion bodies contain ribosomal proteins and ribosome hibernation factors, as well as mRNAs and cognate nascent chains of many proteins in amyloid-like structures, with a bias for membrane proteins with a fold rich in long-range beta-sheet interactions. The peptide is bactericidal against a wide range of pathogenic bacteria in planktonic growth and in biofilms, and reduces bacterial loads in mouse models of Escherichia coli and Acinetobacter baumannii infections. Our results indicate that disrupting protein homeostasis via co-translational aggregation constitutes a promising strategy for development of broad-spectrum antibacterials.
... Finally, mutations in expansion segment 7 of the 25S rRNA gene lead to proteotoxic stress and cause a GFP-VHL reporter protein to form inclusions that also include rRNA and ribosomal proteins [82]. It is conceivable that when ribosome biogenesis is affected, a large number of highly abundant metastable ribosomal proteins and misfolded proteins accumulate due to errors in translation and simply overwhelm the protein homeostasis machinery, resulting in accumulation at the INQ compartment [19,24,82,83]. However, why only a subset of specific proteins accumulate at INQ when ribosome biogenesis is impaired is not clear, and the mechanistic significance of their sequestration remains to be studied. ...
Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with Ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs), Ubp2 and Ubp14, and E3 ligases, Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the Ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the Intranuclear Quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with Ribosomopathies.
... In addition, Lashuel's group [18] reported that the LB formation process is accompanied by the sequestration of cellular factors, including lipids, organelles, and endomembrane structures, which is one of the major drivers of neurodegeneration through disruption of cellular functions and induction of mitochondrial damage and synaptic dysfunctions. As a co-contributor of Alzheimer's disease (AD) with Aβ, the Tau protein aggregates can sequester many other protein factors, including chaperones [28], ribosome components [30,31], and some RBPs [32]. ...
... PQC begins in the ribosome, a protein synthesis machinery. Ribosomal components can be sequestered by the aggregates formed by aggregation-prone proteins, such as Tau [30,31] and polyGR/polyPR (products of mutant C9orf72) [63]. Sequestration of the ribosomal components may cause chronic translation repression and impair protein synthesis. ...
In addition to native-state structures, biomolecules often form condensed supramolecular assemblies or cellular membraneless organelles that are critical for cell life. These biomolecular assemblies, generally including liquid-like droplets (condensates) and amyloid-like aggregates, can sequester or recruit their interacting partners, so as to either modulate various cellular behaviors or even cause disorders. This review article summarizes recent advances in the sequestration of native factors by biomolecular assemblies and discusses their potential consequences on cellular function, homeostasis, and disease pathology.
... Although the exact cellular interactions that contribute to the modulation of neuronal susceptibility still remain largely unknown, the prominent role of cellular proteomic heterogeneity in this process can no longer ignored 19 . Specific protein heterointeractions have been shown to directly influence susceptibility to amyloid formation of several proteins, including among others, Aβ [20][21][22] , tau 23,24 and α-synuclein [25][26][27] , involved in Alzheimer's (AD) and Parkinson's disease (PD), respectively. In the same line, cell-specific inherent metastability of proteins that supersede their solubility levels has been proposed as a generic mechanism that can promote regional protein co-deposition [28][29][30][31][32] . ...
Heterotypic amyloid interactions between related protein sequences have been observed in functional and disease amyloids. While sequence homology seems to favour heterotypic amyloid interactions, we have no systematic understanding of the structural rules determining such interactions nor whether they inhibit or facilitate amyloid assembly. Using structure-based thermodynamic calculations and extensive experimental validation, we performed a comprehensive exploration of the defining role of sequence promiscuity in amyloid interactions. Using tau as a model system we demonstrate that proteins with local sequence homology to tau amyloid nucleating regions can modify fibril nucleation, morphology, assembly and spreading of aggregates in cultured cells. Depending on the type of mutation such interactions inhibit or promote aggregation in a manner that can be predicted from structure. We find that these heterotypic amyloid interactions can result in the subcellular mis-localisation of these proteins. Moreover, equilibrium studies indicate that the critical concentration of aggregation is altered by heterotypic interactions. Our findings suggest a structural mechanism by which the proteomic background can modulate the aggregation propensity of amyloidogenic proteins and we discuss how such sequence-specific proteostatic perturbations could contribute to the selective cellular susceptibility of amyloid disease progression.
... AD patients [78], while additional studies indicate that Reelin impairment also compromises functional downregulation of tau phosphorylation [79]. In another direct example, tau and lysozyme share a common mechanism of induced ribosomal sequestration through co-aggregation which causes dysregulation of protein translation pathways [80,81], while cross-interaction with RNA molecules has been shown to regulate prion aggregation [82,83]. In this line, selective vulnerability to a-synuclein aggregation has been linked with mitochondrial dysfunction and chronic inflammation in PD [84][85][86]. ...
Amyloid aggregation results from the self‐assembly of identical aggregation‐prone sequences into cross‐beta‐sheet structures. The process is best known for its association with a wide range of human pathologies but also as a functional mechanism in all kingdoms of life. Less well elucidated is the role of heterotypic interactions between amyloids and other proteins and macromolecules and how this contributes to disease. We here review current data with a focus on neurodegenerative amyloid‐associated diseases. Evidence indicates that heterotypic interactions occur in a wide range of amyloid processes and that these interactions modify fundamental aspects of amyloid aggregation including seeding, aggregation rates and toxicity. More work is required to understand the mechanistic origin of these interactions, but current understanding suggests that both supersaturation and sequence‐specific binding can contribute to heterotypic amyloid interactions. Further unravelling these mechanisms may help to answer outstanding questions in the field including the selective vulnerability of cells types and tissues and the stereotypical spreading patterns of amyloids in disease.
... The fact, that the intrinsically unstructured tau protein is capable of interacting with polyanionic cofactors like heparin 11 and cellular RNAs 12 , raises the possibility that the tau protein is also capable of interacting with highly charged macromolecular complexes such as the ribosome. Our earlier studies had shown that the partial unfolding or amorphous aggregation of lysozyme and Bovine Carbonic Anhydrase II (BCAII), in the presence of empty non-translating prokaryotic or eukaryotic ribosome, could induce aggregation of ribosomal components 13 . Hence, based on these observations, the present study aims at determining the effect of incubation of the tau protein on the physical integrity of the eukaryotic 80S ribosome. ...
... This method enables us to visualise the total rRNA present in the ribosome as a single consolidated band, as has been used in previous studies on tau-rRNA interactions 24 . In the presence of suitable controls, this method has also been used in our earlier studies as a semi-quantitative technique for following protein-ribosome aggregation process 13 . Similarly, analysis of the protein components of the pellet and the supernatant fractions by denaturing SDS-PAGE was also done which showed that upon incubation of the 80S ribosome with the tau variants K18 and Ht40 (for 6 hours), the presence of ribosomal proteins was observed, both in the supernatant as well as in the pellet (Fig. 2Aiii). ...
... In these experiments the tau variants were incubated with the ribosome in presence of increasing concentrations (0x, 1x, 5x, 10x; x = 0.1 µM) of tRNA or heparin, centrifuged and the aggregation process was followed by agarose gel electrophoresis of rRNA present in the insoluble tau-ribosome aggregates. The highest concentrations of tRNA and heparin used in these experiments were 10-fold higher than that of the 80S ribosome, as was used in our earlier studies 13 . Also, as reported in literature, the cellular stoichiometric ratio of tRNA with respect to the ribosome in yeast is approximately 10-fold 32 . ...
The human tau is a microtubule-associated intrinsically unstructured protein that forms intraneuronal cytotoxic deposits in neurodegenerative diseases, like tauopathies. Recent studies indicate that in Alzheimer’s disease, ribosomal dysfunction might be a crucial event in the disease pathology. Our earlier studies had demonstrated that amorphous protein aggregation in the presence of ribosome can lead to sequestration of the ribosomal components. The present study aims at determining the effect of incubation of the full-length tau protein (Ht40) and its microtubule binding 4-repeat domain (K18) on the eukaryotic ribosome. Our in vitro studies show that incubation of Ht40 and the K18 tau variants with isolated non-translating yeast ribosome can induce a loss of ribosome physical integrity resulting in formation of tau-rRNA-ribosomal protein aggregates. Incubation with the tau protein variants also led to a disappearance of the peak indicating the ribosome profile of the HeLa cell lysate and suppression of translation in the human in vitro translation system. The incubation of tau protein with the ribosomal RNA leads to the formation of tau-rRNA aggregates. The effect of K18 on the yeast ribosome can be mitigated in the presence of cellular polyanions like heparin and tRNA, thereby indicating the electrostatic nature of the aggregation process.
Alzheimer’s disease (AD) is characterized by the appearance of neurofibrillary tangles comprising of the Tau protein and aggregation of amyloid‐β peptides (Aβ 1‐40 and Aβ 1‐42). A concomitant loss of the ribosomal population is also observed in AD‐affected neurons. Our studies demonstrate that, similarly to Tau protein aggregation, in vitro aggregation of Aβ peptides in the vicinity of the yeast 80S ribosome can induce co‐aggregation of ribosomal components. The RNA‐stimulated aggregation of Aβ peptides and the Tau‐K18 variant is dependent on the RNA:protein stoichiometric ratio. A similar effect of stoichiometry is also observed on the ribosome–protein co‐aggregation process. Polyphenolic inhibitors of amyloid aggregation, such as rosmarinic acid and myricetin, inhibit RNA‐stimulated Aβ and Tau‐K18 aggregation and can mitigate the co‐aggregation of ribosomal components. The aggregation of Aβ peptides and the Tau‐K18 variant in the vicinity of the ribosome leads to sequestration of the ribosome. The co‐aggregation of ribosomal components could underlie the loss of neuronal ribosomes in Alzheimer’s disease and resulting neurotoxicity. This process depends upon the ribosome–protein stoichiometry and can be inhibited by amyloid aggregation inhibitors such as rosmarinic acid and myricetin.
To overcome environmental problems caused by the use of fossil resources, microbial cell factories have become a promising technique for the sustainable and eco-friendly development of valuable products from renewable resources. Constructing microbial cell factories with high titers, yields, and productivity requires a balance between growth and production; to this end, tuning gene expression and regulation is necessary to optimise and precisely control complicated metabolic fluxes. In this article, we review the current trends and advances in tuning gene expression and regulation and consider their engineering at each of the three stages of gene regulation: genomic, mRNA, and protein. In particular, the technological approaches utilised in a diverse range of genetic-engineering-based tools for the construction of microbial cell factories are reviewed and representative applications of these strategies are presented. Finally, the prospects for strategies and systems for tuning gene expression and regulation are discussed.
The amyloidoses constitute a group of diseases occurring in humans and animals that are characterized by abnormal deposits of aggregated proteins in organs, affecting their structure and function. In the Abyssinian cat breed, a familial form of renal amyloidosis has been described. In this study, multi-omics analyses were applied and integrated to explore some aspects of the unknown pathogenetic processes in cats. Whole-genome sequences of two affected Abyssinians and 195 controls of other breeds (part of the 99 Lives initiative) were screened to prioritize potential disease-associated variants. Proteome and miRNAome from formalin-fixed paraffin-embedded kidney specimens of fully necropsied Abyssinian cats, three affected and three non-amyloidosis-affected were characterized. While the trigger of the disorder remains unclear, overall, (i) 35,960 genomic variants were detected; (ii) 215 and 56 proteins were identified as exclusive or overexpressed in the affected and control kidneys, respectively; (iii) 60 miRNAs were differentially expressed, 20 of which are newly described. With omics data integration, the general conclusions are: (i) the familial amyloid renal form in Abyssinians is not a simple monogenic trait; (ii) amyloid deposition is not triggered by mutated amyloidogenic proteins but is a mix of proteins codified by wild-type genes; (iii) the form is biochemically classifiable as AA amyloidosis.
