In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver

Ribosomes from a number of prokaryotic and eukaryotic sources (e.g., Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HCl prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured lactate dehydro-genase from rabbit muscle and glucose-6-phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver, The protein-folding activity of E. coli, ribosomes was found to be present in 50s particles and in 23S rRNA. The 30S particle or 16S rRNA did not show any protein-folding activity. The protein-folding activity of 23S rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein-folding activity of 23S rRNA. This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves

The interplay between transcription and mRNA degradation in Saccharomyces cerevisiae

The cellular transcriptome is shaped by both the rates of mRNA synthesis in the nucleus and mRNA degradation in the cytoplasm under a specified condition. The last decade witnessed an exciting development in the field of post-transcriptional regulation of gene expression which underscored a strong functional coupling between the transcription and mRNA degradation. The functional integration is principally mediated by a group of specialized promoters and transcription factors that govern the stability of their cognate transcripts by “marking” them with a specific factor termed “coordinator.” The “mark” carried by the message is later decoded in the cytoplasm which involves the stimulation of one or more mRNA-decay factors, either directly by the “coordinator” itself or in an indirect manner. Activation of the decay factor(s), in turn, leads to the alteration of the stability of the marked message in a selective fashion. Thus, the integration between mRNA synthesis and decay plays a potentially significant role to shape appropriate gene expression profiles during cell cycle progression, cell division, cellular differentiation and proliferation, stress, immune and inflammatory responses, and may enhance the rate of biological evolution

Reactivation of denatured fungal glucose 6-phosphate dehydrogenase and E.coli alkaline phosphatase with E.coli ribosome

Fungal glucose 6-phosphate dehydrogenase and E.coli alkaline phosphatase were denatured either by physical or by chemical means. In vitro reconstitution of these denatured enzymes was assisted by 70S E.coli ribosome, as shown by the recovery of their catalytic competence. Almost total recovery of the activities of completely inactivated enzymes was obtained when 70S ribosome was present at about equimolar concentration with the enzyme molecules at 37C and 50C, respectively

Degradation of Normal mRNA in the Nucleus of Saccharomyces cerevisiae

A nuclear mRNA degradation (DRN) system was identified from analysis of mRNA turnover rates in nup116-Δ strains of Saccharomyces cerevisiae lacking the ability to export all RNAs, including poly(A) mRNAs, at the restrictive temperature. Northern blotting, in situ hybridization, and blocking transcription with thiolutin in nup116-Δ strains revealed a rapid degradation of mRNAs in the nucleus that was suppressed by the rrp6-Δ, rai1-Δ, and cbc1-Δ deletions, but not by the upf1-Δ deletion, suggesting that DRN requires Rrp6p, a 3′-to-5′ nuclear exonuclease, the Rat1p, a 5′-to-3′ nuclear exonuclease, and Cbc1p, a component of CBC, the nuclear cap binding complex, which may direct the mRNAs to the site of degradation. We propose that certain normal mRNAs retained in the nucleus are degraded by the DRN system, similar to degradation of transcripts with 3′ end formation defects in certain mutants

The Role of Nuclear Cap Binding Protein Cbc1p of Yeast in mRNA Termination and Degradation

The cyc1-512 mutation in Saccharomyces cerevisiae causes a 90% reduction in the level of iso-1-cytochrome c because of the lack of a proper 3′-end-forming signal, resulting in low levels of eight aberrantly long cyc1-512 mRNAs which differ in length at their 3′ termini. cyc1-512 can be suppressed by deletion of either of the nonessential genes CBC1 and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex. The upf1-Δ deletion suppressed the cyc1-512 defect by diminishing degradation of the longer subset of cyc1-512 mRNAs, suggesting that downstream elements or structures occurred in the extended 3′ region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations. On the other hand, suppression of cyc1-512 defects by cbc1-Δ occurred by two different mechanisms. The levels of the shorter cyc1-512 transcripts were enhanced in the cbc1-Δ mutants by promoting 3′-end formation at otherwise-weak sites, whereas the levels of the longer cyc1-512 transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation. Furthermore, cbc1-Δ greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1 strain which is defective in mRNA export. We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei

Synthesis of Triazole-Substituted Quinazoline Hybrids for Anticancer Activity and a Lead Compound as the EGFR Blocker and ROS Inducer Agent

A series of triazole-substituted quinazoline hybrid compounds were designed and synthesized for anticancer activity targeting epidermal growth factor receptor (EGFR) tyrosine kinase. Most of the compounds showed moderate to good antiproliferative activity against four cancer cell lines (HepG2, HCT116, MCF-7, and PC-3). Compound 5b showed good antiproliferative activity (IC50 = 20.71 μM) against MCF-7 cell lines. Molecular docking results showed that compound 5b formed hydrogen bond with Met 769 and Lys 721 and π–sulfur interaction with Met 742 of EGFR tyrosine kinase (PDB ID: 1M17). Compound 5b decreases the expression of EGFR and p-EGFR. It also induces apoptosis through reactive oxygen species generation, followed by the change in mitochondrial membrane potential

Binding and conformation of denatured horseradish peroxidase during E. coli ribosome mediated folding

Denatured horseradish peroxidase (HRP) refolded in the presence of intact 70S E. coli ribosome. Fluorescence spectroscopic evidence of direct physical association between the ribosome particles and the denatured HRP during refolding has been detected. The efficiency of energy transfer from the single tryptophan (Trp) to the heme moiety and the quenching patterns of the Trp fluorescence by iodide and acrylamide differed with time while folding in the presence and absence of ribosome. An estimate of the binding of denatured fluorescein-conjugated HRP with ribosome was obtained from polarization measurements (Kd = 41 nM)

Small-Molecule Cdc25A Inhibitors Protect Neuronal Cells from Death Evoked by NGF Deprivation and 6‑Hydroxydopamine

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative diseases that are presently incurable. There have been reports of aberrant activation of cell cycle pathways in neurodegenerative diseases. Previously, we have found that Cdc25A is activated in models of neurodegenerative diseases, including AD and PD. In the present study, we have synthesized a small library of molecules targeting Cdc25A and tested their neuroprotective potential in cellular models of neurodegeneration. The Buchwald reaction and amide coupling were crucial steps in synthesizing the Cdc25A-targeting molecules. Several of these small-molecule inhibitors significantly prevented neuronal cell death induced by nerve growth factor (NGF) deprivation as well as 6-hydroxydopamine (6-OHDA) treatment. Lack of NGF signaling leads to neuron death during development and has been associated with AD pathogenesis. The NGF receptor TrkA has been reported to be downregulated at the early stages of AD, and its reduction is linked to cognitive failure. 6-OHDA, a PD mimic, is a highly oxidizable dopamine analogue that can be taken up by the dopamine transporters in catecholaminergic neurons and can induce cell death by reactive oxygen species (ROS) generation. Some of our newly synthesized molecules inhibit Cdc25A phosphatase activity, block loss of mitochondrial activity, and inhibit caspase-3 activation caused by NGF deprivation and 6-OHDA. Hence, it may be proposed that Cdc25A inhibition could be a therapeutic possibility for neurodegenerative diseases and these Cdc25A inhibitors could be effective treatments for AD and PD