Amyloid protein-induced sequestration of the eukaryotic ribosome: effect of stoichiometry and polyphenolic inhibitors.

Alzheimer's disease (AD) is characterized by the appearance of neurofibrillary tangles comprising of the Tau protein and aggregation of amyloid-ß peptides (Aß 1-40 and Aß 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 Aß peptides in the vicinity of the yeast 80S ribosome can induce co-aggregation of ribosomal components. The RNA-stimulated aggregation of Aß 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 Aß and Tau-K18 aggregation and can mitigate the co-aggregation of ribosomal components.

Amplification of Amyloid Protein-induced Aggregation of the Eukaryotic Ribosome.

BACKGROUND: Alzheimer's disease (AD) is characterized by the aggregation of Tau protein and Amyloid-ß peptides (Aß 1-40 and Aß 1-42). A loss of ribosomal population is also observed in the neurons in affected regions of AD. Our studies demonstrated that in vitro aggregation of amyloid forming proteins, Aß peptides and Tau protein variants (AFPs), in the vicinity of yeast 80S ribosome can induce co-aggregation of ribosomal components. OBJECTIVE: In this study, the ability of minute quantities of AFP-ribosome co-aggregates to seed the aggregation of a large excess of untreated 80S ribosomes was explored. METHODS: The AFPs were purified using ion-exchange chromatography. Seeded aggregation of ribosomes in the presence of minute quantities of ribosome-protein co-aggregates or ribosomal components was studied using agarose gel electrophoretic and SDS-PAGE analysis of the pellets and Sucrose Density Gradient centrifugation of the supernatant obtained after centrifugation of the aggregation reaction mixture. RESULTS: Our studies, therefore, demonstrate that minute quantities of AFP-80S co-aggregate have significant seeding potential and could lead to aggregation of a large excess of fresh 80S ribosomes and this seeding ability is sustained over multiple cycles of ribosome aggregation. The aggregation propensity of ribosomal components alone could contribute towards the seeding of ribosome aggregation. CONCLUSION: The ability of minute quantities of AFP-80S co-aggregates to seed the aggregation of a large excess of fresh 80S ribosomes would result in the loss of global ribosomal population in Alzheimer's disease afflicted neurons. Hence, subject to further validation by in vivo studies, our in vitro studies indicate a significant mode of toxicity of amyloid aggregates that might be important in Alzheimer's disease pathology.

PSRP1 is not a ribosomal protein, but a ribosome-binding factor that is recycled by the ribosome-recycling factor (RRF) and elongation factor G (EF-G).

Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.

Hibernating ribosomes exhibit chaperoning activity but can resist unfolded protein-mediated subunit dissociation.

Ribosome hibernation is a prominent cellular strategy to modulate protein synthesis during starvation and the stationary phase of bacterial cell growth. Translational suppression involves the formation of either factor-bound inactive 70S monomers or dimeric 100S hibernating ribosomal complexes, the biological significance of which is poorly understood. Here, we demonstrate that the Escherichia coli 70S ribosome associated with stationary phase factors hibernation promoting factor or protein Y or ribosome-associated inhibitor A and the 100S ribosome isolated from both Gram-negative and Gram-positive bacteria are resistant to unfolded protein-mediated subunit dissociation and subsequent degradation by cellular ribonucleases. Considering that the increase in cellular stress is accompanied by accumulation of unfolded proteins, such resistance of hibernating ribosomes towards dissociation might contribute to their maintenance during the stationary phase. Analysis of existing structures provided clues on the mechanism of inhibition of the unfolded protein-mediated disassembly in case of hibernating factor-bound ribosome. Further, the factor-bound 70S and 100S ribosomes can suppress protein aggregation and assist in protein folding. The chaperoning activity of these ribosomes is the first evidence of a potential biological activity of the hibernating ribosome that might be crucial for cell survival under stress conditions.

Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins.

Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a three-dimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes. Fitting of the conserved portions of the x-ray crystallographic structure of the bacterial 70S ribosome into our cryo-EM map of the chloro-ribosome reveals the positions of PRP extensions and the locations of the PSRPs. Surprisingly, PSRP1 binds in the decoding region of the small (30S) ribosomal subunit, in a manner that would preclude the binding of messenger and transfer RNAs to the ribosome, suggesting that PSRP1 is a translation factor rather than a ribosomal protein. PSRP2 and PSRP3 appear to structurally compensate for missing segments of the 16S rRNA within the 30S subunit, whereas PSRP4 occupies a position buried within the head of the 30S subunit. One of the two PSRPs in the large (50S) ribosomal subunit lies near the tRNA exit site. Furthermore, we find a mass of density corresponding to chloro-ribosome recycling factor; domain II of this factor appears to interact with the flexible C-terminal domain of PSRP1. Our study provides evolutionary insights into the structural and functional roles that the PSRPs play during protein synthesis in chloroplasts.

Tau protein- induced sequestration of the eukaryotic ribosome: Implications in neurodegenerative disease.

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.

Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA.

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 subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity.

Unfolded protein exhibits antiassociation activity toward the 50S subunit facilitating 70S ribosome dissociation.

The ability of the ribosome to assist in the folding of proteins both in vitro and in vivo is well documented. The interaction of an unfolded protein with the peptidyltransferase center of the bacterial large ribosomal subunit is followed by release of the protein in a folding-competent state and rapid dissociation of ribosome into its subunits. Our studies demonstrate that the 50S subunit-associated antiassociation ability of an unfolded protein might contribute significantly to its ability to mediate energy-independent and stable dissociation of the ribosome into its subunits. The stoichiometry of the protein present with respect to the ribosome is an important factor in determining whether the ribosome has a chaperoning effect on protein folding or if the protein acts as a 50S subunit antiassociation factor. Sustained interaction of the protein with the ribosome at higher protein concentrations and the hindrance in the formation of the central intersubunit bridge B2a could underlie the antiassociation activity of unfolded proteins. The ribosome dissociation and antiassociation activity of unfolded proteins could make the ribosome susceptible to cellular ribonucleases, thereby initiating ribosome degradation, which is a well-documented phenomenon under nutrient deprivation conditions.

Impact of P-Site tRNA and antibiotics on ribosome mediated protein folding: studies using the Escherichia coli ribosome.

BACKGROUND: The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3' -CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity. RESULTS: The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3'-CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability. CONCLUSION: Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.

The ribosome can prevent aggregation of partially folded protein intermediates: studies using the Escherichia coli ribosome.

BACKGROUND: Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone 'foldases' that are distinct from chaperone' holdases' that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC) located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA) is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains. RESULTS: We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII) and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein. CONCLUSION: The ribosome can behave like a 'holdase' chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.

Progression of the ribosome recycling factor through the ribosome dissociates the two ribosomal subunits.

After the termination step of translation, the posttermination complex (PoTC), composed of the ribosome, mRNA, and a deacylated tRNA, is processed by the concerted action of the ribosome-recycling factor (RRF), elongation factor G (EF-G), and GTP to prepare the ribosome for a fresh round of protein synthesis. However, the sequential steps of dissociation of the ribosomal subunits, and release of mRNA and deacylated tRNA from the PoTC, are unclear. Using three-dimensional cryo-electron microscopy, in conjunction with undecagold-labeled RRF, we show that RRF is capable of spontaneously moving from its initial binding site on the 70S Escherichia coli ribosome to a site exclusively on the large 50S ribosomal subunit. This movement leads to disruption of crucial intersubunit bridges and thereby to the dissociation of the two ribosomal subunits, the central event in ribosome recycling. Results of this study allow us to propose a model of ribosome recycling.

Interaction of Era with the 30S ribosomal subunit implications for 30S subunit assembly.

Era (E. coliRas-like protein) is a highly conserved and essential GTPase in bacteria. It binds to the 16S ribosomal RNA (rRNA) of the small (30S) ribosomal subunit, and its depletion leads to accumulation of an unprocessed precursor of the 16S rRNA. We have obtained a three-dimensional cryo-electron microscopic map of the Thermus thermophilus 30S-Era complex. Era binds in the cleft between the head and platform of the 30S subunit and locks the subunit in a conformation that is not favorable for association with the large (50S) ribosomal subunit. The RNA binding KH motif present within the C-terminal domain of Era interacts with the conserved nucleotides in the 3' region of the 16S rRNA. Furthermore, Era makes contact with several assembly elements of the 30S subunit. These observations suggest a direct involvement of Era in the assembly and maturation of the 30S subunit.

Properties of human vasopressin precursor constructs: inefficient monomer folding in the absence of copeptin as a potential contributor to diabetes insipidus.

These studies were aimed at an initial characterization of the human vasopressin precursor and the evaluation of factors leading to misfolding by the pathological 87STOP mutation. This mutation deletes the precursor's glycosylated copeptin segment, which has been considered unnecessary for folding, and the last seven neurophysin residues. We investigated the role in folding of the last seven neurophysin residues by comparing the properties of the 87STOP precursor and its derivative neurophysin with those of the corresponding wild-type proteins from which copeptin had been deleted, leading to the following conclusions. First, despite modulating effects on several protein properties, the last seven neurophysin residues do not make a significant net thermodynamic contribution to precursor folding; stabilities of the mutant and wild-type precursors to both guanidine denaturation and redox buffer unfolding are similar, as are in vitro folding rates. Second, the monomeric forms of both precursors are unstable and predicted to fold inefficiently at physiological pH and temperature, as evidenced by precursor behavior in redox buffers and by thermodynamic calculations. Third, both precursors are significantly less stable than the bovine oxytocin precursor. These results, together with earlier studies elsewhere of vasopressin precursor behavior within rat neurons, are shown to represent a self-consistent argument for a role for glycosylated copeptin in vasopressin precursor folding in vivo, copeptin most probably assisting refolding by facilitating interaction of misfolded monomers with the calnexin/calreticulin system. This hypothesis provides an explanation for the absence of copeptin in the more stable oxytocin precursor and suggests that the loss of copeptin contributes to 87STOP pathogenicity. Reported cell culture studies of rat precursor folding are also discussed in this context. Most generally, the results emphasize the significance of monomer stability in the folding pathways of oligomeric proteins.