Molecular characterization and expression analysis of a QM protein gene in Chinese mitten crab Eriocheir sinensis.

QM protein was previously discovered as a tumor suppressor, and numerous studies have shown that QM protein also played important roles in the immune responses. To investigate the potential roles of the QM protein gene in Eriocheir sinensis, the QM protein gene (designated as EsQM) has been cloned from E. sinensis using the rapid amplification of cDNA ends (RACE) technique. The cDNA of EsQM is 781 bp in length, consisting of a 654 bp open reading frame (ORF), encoding 219 amino acids, a 27 bp 5' untranslated region (UTR) and a 94 bp 3' UTR. The EsQM protein has a calculated molecular weight of 25.4 kDa and a theoretical isoelectric point of 10.10. The deduced protein sequence of EsQM contains a Ribosomal_L16 domain, an SH3-binding motif, an N-acylation site, two putative antibiotic binding sites, two putative protein kinase C phosphorylation sites, and two amidation sites. EsQM is extremely conserved and exhibits more than 85% similarities to previously identified arthropod QM protein genes. By real-time quantitative PCR (qPCR) analysis, we found that EsQM mRNA transcripts were detectable in all the examined tissues, with the highest expression in hemocytes. The mRNA expression of EsQM in hemocytes was significantly upregulated after the stimulation of Aeromonas hydrophila or polybrominated diphenyl ether-47 (BDE-47). Moreover, EsQM mRNA expression in hemocytes responded more quickly and lasted longer when stimulated by A.hydrophila than BDE-47. Thus, EsQM can respond to bacterial infection and environmental pollution, and might be involved in the defense mechanism to both biological and non-biological stimulation of arthropods.

The ufmylation modification of ribosomal protein L10 in the development of pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is the most malignant cancer with a high mortality rate. Despite the association of ribosomal protein L10 (RPL10) with PAAD and previous reports on RPL26 ufmylation, the relationship between RPL10 ufmylation and PAAD development remains unexplored. Here, we report the dissection of ufmylating process of RPL10 and potential roles of RPL10 ufmylation in PAAD development. The ufmylation of RPL10 was confirmed in both pancreatic patient tissues and cell lines, and specific modification sites were identified and verified. Phenotypically, RPL10 ufmylation significantly increased cell proliferation and stemness, which is principally resulted from higher expression of transcription factor KLF4. Moreover, the mutagenesis of ufmylation sites in RPL10 further demonstrated the connection of RPL10 ufmylation with cell proliferation and stemness. Collectively, this study reveals that PRL10 ufmylation plays an important role to enhance the stemness of pancreatic cancer cells for PAAD development.

Hybrid QM/MM Simulations Confirm Zn(II) Coordination Sphere That Includes Four Cysteines from the P2 × 4R Head Domain.

To ascertain the role of Zn(II) as an allosteric modulator on P2X4R, QM/MM molecular dynamic simulations were performed on the WT and two P2X4R mutants suggested by previous electrophysiological data to affect Zn(II) binding. The Gibbs free energy for the reduction of the putative P2X4R Zn(II) binding site by glutathione was estimated at -22 kcal/mol. Simulations of the WT P2X4R head domain revealed a flexible coordination sphere dominated by an octahedral geometry encompassing C126, N127, C132, C149, C159 and a water molecule. The C132A mutation disrupted the metal binding site, leading to a coordination sphere with a majority of water ligands, and a displacement of the metal ion towards the solvent. The C132A/C159A mutant exhibited a tendency towards WT-like stability by incorporating the R148 backbone to the coordination sphere. Thus, the computational findings agree with previous experimental data showing Zn(II) modulation for the WT and C132A/C159A variants, but not for the C132A mutant. The results provide molecular insights into the nature of the Zn(II) modulation in P2X4R, and the effect of the C132A and C132A/C159A mutations, accounting for an elusive modulation mechanism possibly occurring in other extracellular or membrane protein.

Interaction with Ribosomal Proteins Accompanies Stress Induction of the Anticancer Metallodrug BOLD-100/KP1339 in the Endoplasmic Reticulum.

The ruthenium-based anticancer agent BOLD-100/KP1339 has shown promising results in several in vitro and in vivo tumour models as well as in early clinical trials. However, its mode of action remains to be fully elucidated. Recent evidence identified stress induction in the endoplasmic reticulum (ER) and concomitant down-modulation of HSPA5 (GRP78) as key drug effects. By exploiting the naturally formed adduct between BOLD-100 and human serum albumin as an immobilization strategy, we were able to perform target-profiling experiments that revealed the ribosomal proteins RPL10, RPL24, and the transcription factor GTF2I as potential interactors of this ruthenium(III) anticancer agent. Integrating these findings with proteomic profiling and transcriptomic experiments supported ribosomal disturbance and concomitant induction of ER stress. The formation of polyribosomes and ER swelling of treated cancer cells revealed by TEM validated this finding. Thus, the direct interaction of BOLD-100 with ribosomal proteins seems to accompany ER stress-induction and modulation of GRP78 in cancer cells.

Ribosomal Protein L10: From Function to Dysfunction.

Eukaryotic cytoplasmic ribosomes are highly structured macromolecular complexes made up of four different ribosomal RNAs (rRNAs) and 80 ribosomal proteins (RPs), which play a central role in the decoding of genetic code for the synthesis of new proteins. Over the past 25 years, studies on yeast and human models have made it possible to identify RPL10 (ribosomal protein L10 gene), which is a constituent of the large subunit of the ribosome, as an important player in the final stages of ribosome biogenesis and in ribosome function. Here, we reviewed the literature to give an overview of the role of RPL10 in physiologic and pathologic processes, including inherited disease and cancer.

Ribosomal protein QM/RPL10 positively regulates defence and protein translation mechanisms during nonhost disease resistance.

Ribosomes play an integral part in plant growth, development, and defence responses. We report here the role of ribosomal protein large (RPL) subunit QM/RPL10 in nonhost disease resistance. The RPL10-silenced Nicotiana benthamiana plants showed compromised disease resistance against nonhost pathogen Pseudomonas syringae pv. tomato T1. The RNA-sequencing analysis revealed that many genes involved in defence and protein translation mechanisms were differentially affected due to silencing of NbRPL10. Arabidopsis AtRPL10 RNAi and rpl10 mutant lines showed compromised nonhost disease resistance to P. syringae pv. tomato T1 and P. syringae pv. tabaci. Overexpression of AtRPL10A in Arabidopsis resulted in reduced susceptibility against host pathogen P. syringae pv. tomato DC3000. RPL10 interacts with the RNA recognition motif protein and ribosomal proteins RPL30, RPL23, and RPS30 in the yeast two-hybrid assay. Silencing or mutants of genes encoding these RPL10-interacting proteins in N. benthamiana or Arabidopsis, respectively, also showed compromised disease resistance to nonhost pathogens. These results suggest that QM/RPL10 positively regulates the defence and translation-associated genes during nonhost pathogen infection.

Soma-Targeted Imaging of Neural Circuits by Ribosome Tethering.

Neuroscience relies on techniques for imaging the structure and dynamics of neural circuits, but the cell bodies of individual neurons are often obscured by overlapping fluorescence from axons and dendrites in surrounding neuropil. Here, we describe two strategies for using the ribosome to restrict the expression of fluorescent proteins to the neuronal soma. We show first that a ribosome-tethered nanobody can be used to trap GFP in the cell body, thereby enabling direct visualization of previously undetectable GFP fluorescence. We then design a ribosome-tethered GCaMP for imaging calcium dynamics. We show that this reporter faithfully tracks somatic calcium dynamics in the mouse brain while eliminating cross-talk between neurons caused by contaminating neuropil. In worms, this reporter enables whole-brain imaging with faster kinetics and brighter fluorescence than commonly used nuclear GCaMPs. These two approaches provide a general way to enhance the specificity of imaging in neurobiology.

Nuclei of HeLa cells interactomes unravel a network of ghost proteins involved in proteins translation.

Ghost proteins are issued from alternative Open Reading Frames (ORFs) and are missing a genome annotation. Indeed, historical filters applied for the detection of putative translated ORFs led to a wrong classification of transcripts considered as non-coding although translated proteins can be detected by proteomics. This Ghost (also called Alternative) proteome was neglected, and one major issue is to identify the implication of the Ghost proteins in the biological processes. In this context, we aimed to identify the protein-protein interactions (PPIs) of the Ghost proteins. For that, we re-explored a cross-link MS study performed on nuclei of HeLa cells using cross-linking mass spectrometry (XL-MS) associated with the HaltOrf database. Among 1679 cross-link interactions identified, 292 are involving Ghost Proteins. Forty-Four of these Ghost proteins are found to interact with 7 Reference proteins related to ribonucleoproteins, ribosome subunits and zinc finger proteins network. We, thus, have focused our attention on the heterotrimer between the RE/poly(U)-binding/degradation factor 1 (AUF1), the Ribosomal protein 10 (RPL10) and AltATAD2. Using I-Tasser software we performed docking models from which we could suggest the attachment of AUF1 on the external part of RPL10 and the interaction of AltATAD2 on the RPL10 region interacting with 5S ribosomal RNA as a mechanism of regulation of the ribosome. Taken together, these results reveal the importance of Ghost Proteins within known protein interaction networks.

A QM protein from Bombyx mori negatively regulates prophenoloxidase activation and melanization by interacting with Jun protein.

The QM gene that encodes for the ribosomal protein L10 was firstly identified from human tumour cells as a tumour suppressor. In this study, a QM gene was identified in silkworm Bombyx mori (BmQM) and its immunomodulatory function was explored. BmQM messenger RNA (mRNA) and protein were highly expressed in the silk gland and fat body, and expressed in all stages of silkworm growth. After challenged with four different microorganisms, the expression levels of BmQM mRNA in fat body or haemocytes were significantly upregulated compared with the control. After knock-down of BmQM gene, the expressions of some immune genes (PGRPS6, Gloverin0, Lysozyme and Moricin) were affected, and the transcripts of prophenoloxidase1 and prophenoloxidase2 have different degrees of change. The phenoloxidase activity was significantly reduced when the purified recombinant BmQM protein was injected. Recombinant BmQM protein inhibited systemic melanization and suppressed prophenoloxidase activation stimulated by Micrococcus luteus, but it did not affect phenoloxidase activity. Far-western blotting assays showed that the BmQM protein interacted with silkworm BmJun protein, which negatively regulates AP-1 expression. Our results indicated that BmQM protein could affect some immune gene expression and negatively regulate the prophenoloxidase-activating system, and it may play an important role in regulation of the innate immunity in insects.