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.

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.

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.

Rhinosporidiosis of the left wrist joint: a case report.

Rhinosporidiosis is a chronic granulomatous disease caused by Rhinosporidium seeberi. It usually occurs in the mucous membranes of nose, nasopharynx, and eyes, and less commonly in extra nasal sites such as skin, bones, genitalia, and even the internal organs. Rhinosporidiosis occurs in the wrist joint with isolated bony involvement is rare. We report one such case in a 50-year-old man who presented with a non-tender, fixed swelling over his anterolateral aspect of left forearm. Radiography and computed tomography showed a lytic destructive lesion involving the distal radius, ulna, carpals, and base of metacarpals. Biopsy revealed features of rhinosporidiosis. The patient underwent below-elbow amputation.