Ribosome-DnaK interactions in relation to protein folding

Bacterial ribosomes or their 50S subunit can refold many unfolded proteins. The folding activity resides in domain V of 23S RNA of the 50S subunit. Here we show that ribosomes can also refold a denatured chaperone, DnaK, in vitro, and the activity may apply in the folding of nascent DnaK polypeptides in vivo. The chaperone was unusual as the native protein associated with the 50S subunit stably with a 1:1 stoichiometry in vitro. The binding site of the native protein appears to be different from the domain V of 23S RNA, the region with which denatured proteins interact. The DnaK binding influenced the protein folding activity of domain V modestly. Conversely, denatured protein binding to domain V led to dissociation of the native chaperone from the 50S subunit. DnaK thus appears to depend on ribosomes for its own folding, and upon folding, can rebind to ribosome to modulate its general protein folding activity

Role of the ribosome in protein folding

In all organisms, the ribosome synthesizes and folds full length polypeptide chains into active three-dimensional conformations. The nascent protein goes through two major interactions, first with the ribosome which synthesizes the polypeptide chain and holds it for a considerable length of time, and then with the chaperones. Some of the chaperones are found in solution as well as associated to the ribosome. A number of in vitro and in vivo experiments revealed that the nascent protein folds through specific interactions of some amino acids with the nucleotides in the peptidyl transferase center (PTC) in the large ribosomal subunit. The mechanism of this folding differs from self-folding. In this article, we highlight the folding of nascent proteins on the ribosome and the influence of chaperones etc. on protein folding

Molnupiravir or nirmatrelvir-ritonavir versus usual care in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Background: Molnupiravir and nirmatrelvir-ritonavir (Paxlovid) are oral antivirals that have been proposed as treatments for patients admitted to hospital with COVID-19. Methods: In this randomised, controlled, open-label, adaptive platform trial, several potential treatments for patients hospitalised with COVID-19 pneumonia were evaluated. Molnupiravir and nirmatrelvir-ritonavir were assessed in separate comparisons in RECOVERY, both of which are reported here. Eligible and consenting adults could join the molnupiravir comparison, the nirmatrelvir-ritonavir comparison, or both. For each comparison, participants were randomly allocated in a 1:1 ratio to the relevant antiviral (five days of molnupiravir 800mg twice daily or nirmatrelvir-ritonavir 300mg/100mg twice daily) or to usual care without the relevant antiviral drug, using web-based unstratified randomisation with allocation concealment. The primary outcome was 28-day mortality, and secondary outcomes were time to discharge alive from hospital, and among those not on invasive ventilation at baseline, progression to invasive ventilation or death. Analysis was by intention-to-treat. Both comparisons were stopped by the investigators because of low recruitment. ISRCTN (50189673) and clinicaltrials.gov (NCT04381936). Findings: From 24 January 2022 to 24 May 2023, 923 patients were recruited to the molnupiravir comparison (445 allocated molnupiravir and 478 allocated usual care), and from 31 March 2022 to 24 May 2023, 137 patients were recruited to the nirmatrelvir-ritonavir comparison (68 allocated nirmatrelvir-ritonavir and 69 allocated usual care). More than three-quarters of the patients in both comparisons were vaccinated and had anti-spike antibodies at randomisation, and more than two-thirds were receiving other SARS-CoV-2 antivirals (including remdesivir or sotrovimab). In the molnupiravir comparison, 74 (17%) patients allocated to molnupiravir and 79 (17%) patients allocated usual care died within 28 days (hazard ratio [HR] 0.93; 95% confidence interval [CI] 0.68-1.28; p=0.66). In the nirmatrelvir-ritonavir comparison, 13 (19%) patients allocated nirmatrelvir-ritonavir and 13 (19%) patients allocated usual care died within 28 days (HR 1.02; 95% CI 0.47-2.23; p=0.96). In neither comparison was there evidence of a significant difference in the duration of hospitalisation or the proportion of patients progressing to invasive ventilation or death. Interpretation: In adults hospitalised with COVID-19, neither molnupiravir nor nirmatrelvir-ritonavir were associated with reductions in 28-day mortality, duration of hospital stay, or risk of progressing to invasive mechanical ventilation or death although these comparisons had limited statistical power due to low recruitment. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health and Care Research (Grant ref: MC_PC_19056), and Wellcome Trust (Grant Ref: 222406/Z/20/Z). Trial registration: ClinicalTrials.gov NCT04381936 https://clinicaltrials.gov/ct2/show/NCT0438193

Growth, cell division and sporulation in mycobacteria

Bacteria have the ability to adapt to different growth conditions and to survive in various environments. They have also the capacity to enter into dormant states and some bacteria form spores when exposed to stresses such as starvation and oxygen deprivation. Sporulation has been demonstrated in a number of different bacteria but Mycobacterium spp. have been considered to be non-sporulating bacteria. We recently provided evidence that Mycobacterium marinum and likely also Mycobacterium bovis bacillus Calmette–Guérin can form spores. Mycobacterial spores were detected in old cultures and our findings suggest that sporulation might be an adaptation of lifestyle for mycobacteria under stress. Here we will discuss our current understanding of growth, cell division, and sporulation in mycobacteria

One-pot synthesis of chromeno[2,3-<i>b</i>]isoindolo[1,2-<i>e</i>]pyrrole-12,13-dione derivatives by sequential reaction of ninhydrin, 2-aminochromen-4-ones and arylamines

<p>Stirring an equimolar mixture of ninhydrin <b>1</b> and 2-aminochromen-4-ones <b>2</b> in CH₃COOH at room temperature produced 6a,11a-dihydroxy-6<i>H</i>-chromeno[2,3-<i>b</i>]indeno[2,1-<i>d</i>]pyrrole-11,12(6a<i>H</i>,11a<i>H</i>)-diones <b>3</b>, which on heating with aromatic amines <b>6</b> in acetic acid produced 11b-hydroxy-7-<i>N</i>-arylimino-6<i>H</i>-chromeno[2,3-<i>b</i>]isoindolo[1,2-<i>e</i>]pyrrole-12,13(11b<i>H</i>)-diones <b>7</b>.</p

Nickel (II) complexes incorporating pyridyl, imine and amino chelate ligands: synthesis, structure, isomer preference, structural transformation and reactivity towards nickel (III) derivatives

Facile condensation of 2-acetylpyridine with ethylenediamine in a 1:1 or 2:1 molar ratio yielded two neutral ligands with different denticity: 1-amino-4-(2-pyridyl)-3-azapent-3-ene (tridentate, L1) and 2,7-bis(2-pyridyl)-3,6-diazaocta-2,6-diene (tetradentate, L3), respectively. Replacing the ketone with 2-pyridinecarboxaldehyde gave a similar set of condensates (L2 and L4). The tridentate mono-Schiff bases (L1 and L2) react smoothly with Ni(ClO4)2•6H2O furnishing the brown bis-chelate complexes (1a and 1b) with an NiN6 coordination sphere, while the tetradentate bis-Schiff bases (L3 and L4) form green pseudo-octahedral complexes (2a and 2b) in which NiII is present in an N4O2 coordination environment. The isomer specificity for both types of complexes is conspicuous from the representative X-ray structures of 1a and 2a. cis-trans-cis and cis-cis-trans isomers are found exclusively for 1 and 2, respectively. The crystal structure of 2a reveals O−H•••O hydrogen bond interactions assembling alternating cations and anions in an infinite chain-like array. Cyclic voltammetric measurements of 1 and 2 in MeCN solution show a quasi-reversible one-electron oxidation near 0.95 and 0.87 V (vs. SCE), respectively, attributed to a NiIII-NiII redox couple. Another irreversible NiIV-NiIII redox response was observed at higher potential near 1.70 and 1.80 V (vs. SCE) for 1 and 2, respectively. Complexes 1 and 2 display a weak, broad d-d transition band along with a charge-transfer transition. Magnetic susceptibility measurements (at 298 K) confirmed that the spin states of the NiII centres in 1 and 2 are same, S = 1, in agreement with an octahedral configuration. Complexes 1 form stable reddish-brown NiIII complexes (3) under exhaustive constant-potential electrolysis treatment. The NiIII complexes display axial EPR spectra both at 298 K and 77 K with g&#8869; &#62; g|| indicating the presence of an unpaired electron primarily on the metal centre. Complexes of type 1 and 3 exhibit virtually superimposable cyclic voltammograms in a reverse electrode scan study which confirms no change in the coordination sphere on going from 1 to 3. The effective magnetic moment values (ca. 2.10 &#956;B) of 3 suggest a significant orbital contribution to the paramagnetism, consistent with a tetragonally distorted low spin 3d7 system. A colour change phenomenon has been observed for type 2 complexes

MAL expression downregulation through suppressive H3K27me3 marks at the promoter in HPV16-related cervical cancers is prognostically relevant and manifested by the interplay of novel MAL antisense long noncoding RNA AC103563.8, E7 oncoprotein and EZH2

Abstract Background MAL (T-lymphocyte maturation-associated protein) is highly downregulated in most cancers, including cervical cancer (CaCx), attributable to promoter hypermethylation. Long noncoding RNA genes (lncGs) play pivotal roles in CaCx pathogenesis, by interacting with human papillomavirus (HPV)-encoded oncoproteins, and epigenetically regulating coding gene expression. Hence, we attempted to decipher the impact and underlying mechanisms of MAL downregulation in HPV16-related CaCx pathogenesis, by interrogating the interactive roles of MAL antisense lncRNA AC103563.8, E7 oncoprotein and PRC2 complex protein, EZH2. Results Employing strand-specific RNA-sequencing, we confirmed the downregulated expression of MAL in association with poor overall survival of CaCx patients bearing HPV16, along with its antisense long noncoding RNA (lncRNA) AC103563.8. The strength of positive correlation between MAL and AC103563.8 was significantly high among patients compared to normal individuals. While downregulated expression of MAL was significantly associated with poor overall survival of CaCx patients bearing HPV16, AC103563.8 did not reveal any such association. We confirmed the enrichment of chromatin suppressive mark, H3K27me3 at MAL promoter, using ChIP-qPCR in HPV16-positive SiHa cells. Subsequent E7 knockdown in such cells significantly increased MAL expression, concomitant with decreased EZH2 expression and H3K27me3 marks at MAL promoter. In silico analysis revealed that both E7 and EZH2 bear the potential of interacting with AC103563.8, at the same binding domain. RNA immunoprecipitation with anti-EZH2 and anti-E7 antibodies, respectively, and subsequent quantitative PCR analysis in E7-silenced and unperturbed SiHa cells confirmed the interaction of AC103563.8 with EZH2 and E7, respectively. Apparently, AC103563.8 seems to preclude EZH2 and bind with E7, failing to block EZH2 function in patients. Thereby, enhanced EZH2 expression in the presence of E7 could potentially inactivate the MAL promoter through H3K27me3 marks, corroborating our previous results of MAL expression downregulation in patients. Conclusion AC103563.8-E7-EZH2 axis, therefore, appears to crucially regulate the expression of MAL, through chromatin inactivation in HPV16-CaCx pathogenesis, warranting therapeutic strategy development

Overexpression of EGFR in head and neck squamous cell carcinoma is associated with inactivation of SH3GL2 and CDC25A genes.

The aim of this study is to understand the mechanism of EGFR overexpression in head and neck squamous cell carcinoma (HNSCC). For this reason, expression/mutation of EGFR were analyzed in 30 dysplastic head and neck lesions and 148 HNSCC samples of Indian patients along with 3 HNSCC cell lines. In addition, deletion/methylation/mutation/expression of SH3GL2 (associated with EGFR degradation) and CDC25A (associated with dephosphorylation of EGFR) were analyzed in the same set of samples. Our study revealed high frequency of EGFR overexpression (66-84%), low frequency of gene amplification (10-32.5%) and absence of functional mutation in the dysplastic lesions and HNSCC samples. No correlation was found between protein overexpression and mRNA expression/gene amplification status of EGFR. On the other hand, frequent alterations (deletion/methylation) of SH3GL2 (63-77%) and CDC25A (37-64%) were seen in the dysplastic and HNSCC samples. Two novel single nucleotide polymorphism (SNPs) were found in the promoter region of SH3GL2. Reduced expression of these genes showed concordance with their alterations. Overexpression of EGFR and p-EGFR were significantly associated with reduced expression and alterations of SH3GL2 and CDC25A respectively. In-vitro demethylation experiment by 5-aza-2'-deoxycytidine (5-aza-dC) showed upregulation of SH3GL2 and CDC25A and downregulation of EGFR expression in Hep2 cell line. Poor patient outcome was predicted in the cases with alterations of SH3GL2 and CDC25A in presence of human papilloma virus (HPV) infection. Also, low SH3GL2 and high EGFR expression was a predictor of poor patient survival. Thus, our data suggests that overexpression of EGFR due to its reduced degradation and dephosphorylation is needed for development of HNSCC