Ribosomal Protein L11 Research Articles

Probing the antioxidant activity of functional proteins and bioactive peptides in Hermetia illucens larvae fed with food wastes

Food waste is becoming more prevalent, and managing it is one of the most important issues in terms of food safety. In this study, functional proteins and bioactive peptides produced from the enzymatic digestion of black soldier fly (Hermetia illucens L., BSF) fed with food wastes were characterized and quantified using proteomics-based analysis. The results revealed approximately 78 peptides and 57 proteins, including 40S ribosomal protein S4, 60S ribosomal protein L8, ATP synthase subunit alpha, ribosomal protein S3, Histone H2A, NADP-glutamate dehydrogenase, Fumarate hydratase, RNA helicase, Chitin binding Peritrophin-A, Lectin C-type protein, etc. were found in BSF. Furthermore, functional analysis of the proteins revealed that the 60S ribosomal protein L5 (RpL5) in BSF interacted with a variety of ribosomal proteins and played a key role in the glycolytic process (AT14039p). Higher antioxidant activity was found in peptide sequences such as GYGFGGGAGCLSMDTGAHLNR, VVPSANRAMVGIVAGGGRIDKPILK, AGLQFPVGR, GFKDQIQDVFK, and GFKDQIQDVFK. It was concluded that the bioconversion of food wastes by BSF brought about the generation of a variety of functional proteins and bioactive peptides with strong antioxidant activity. However, more studies are required to exploit BSF's potential in the value addition of food wastes.

Caenorhabditis elegans Parkin: Regulators of its abundance and role in autophagy-lysosomal dynamics

Background: Parkin, which when mutated leads to early-onset Parkinson’s disease, acts as an E3 ubiquitin ligase. How Parkin is regulated for selective protein and organelle targeting is not well understood. Here, we used protein interactor and genetic screens in Caenorhabditis elegans (C. elegans) to identify new regulators of Parkin abundance and showed their impact on autophagy-lysosomal dynamics and alpha-Synuclein processing. Methods: We generated a transgene encoding mCherry-tagged C. elegans Parkin – Parkinson’s Disease Related 1 (PDR-1). We performed protein interactor screen using Co-immunoprecipitation followed by mass spectrometry analysis to identify putative interacting partners of PDR-1. Ribonucleic acid interference (RNAi) screen and an unbiased mutagenesis screen were used to identify genes regulating PDR-1 abundance. Confocal microscopy was used for the identification of the subcellular localization of PDR-1 and alpha-Synuclein processing. Results: We show that the mCherry::pdr-1 transgene rescues the mitochondrial phenotype of pdr-1 mutants and that the expressed PDR-1 reporter is localized in the cytosol with enriched compartmentalization in the autophagy-lysosomal system. We determined that the transgenic overexpression of the PDR-1 reporter, due to inactivated small interfering RNA (siRNA) generation pathway, disrupts autophagy-lysosomal dynamics. From the RNAi screen of putative PDR-1 interactors we found that the inactivated Adenine Nucleotide Translocator ant-1.1/hANT, or hybrid ubiquitin genes ubq-2/hUBA52 and ubl-1/hRPS27A encoding a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively, induced PDR-1 abundance and affected lysosomal dynamics. In addition, we demonstrate that the abundant PDR-1 plays a role in alpha-Synuclein processing. Conclusions: These data show that the abundant C. elegans Parkin ortholog affects the autophagy-lysosomal system together with alpha-Synuclein processing which can help in understanding the pathology in Parkin-related diseases.

Direct and indirect roles of GRWD1 in the inactivation of p53 in cancer.

Glutamate-rich WD40 repeat containing 1 (GRWD1), also known as WDR28, interacts with various proteins through its WD domain and is involved in transcription, translation, cell cycle progression, ubiquitin-mediated degradation and DNA replication and repair. Ribosomal protein L11 (RPL11), which directly interacts with MDM2, inhibits MDM2 ubiquitin ligase activity, thus promoting p53 stabilization. Binding of GRWD1 to RPL11 disrupts the interaction between RPL11 and MDM2 and promotes p53 ubiquitination by MDM2. In addition, a recent report by Fujiyama et al. found that GRWD1 also directly interacts with wild-type p53 and suppresses its transcriptional activity. They propose that GRWD1 is a novel tumor-promoting molecule that negatively regulates wild-type p53 via both indirect and direct mechanisms.

Proteomic Profiles of Staphylococcus aureus Strains Associated with Subclinical Bovine Mastitis.

Staphylococcus aureus is the main pathogen associated with bovine mastitis, an intramammary inflammation that leads to significant economic losses in dairy herds. Efforts have been made to identify the bacterial determinants important to the infective process but most of the studies are focused on surface and secreted proteins. Considering that virulence is affected by metabolism, in this study we contrasted the proteome of strains of S. aureus causing persistent subclinical (Sau302 and Sau340) and clinical bovine mastitis (RF122). Protein expressions from cytosolic fractions of bacteria grown under conditions mimicking the mastitic mammary glands are reported. A total of 342 proteins was identified, 52 of which were differentially expressed. Among those down-regulated in the subclinical strains were the two-component sensor histidine kinase SaeS and PurH, both involved in bacterial virulence. The ribosome hibernation promotion factor and the 50S ribosomal protein L13 were up-regulated suggesting that Sau302 and Sau340 modulate protein translation, a condition that may contribute to bacterial survival under stressful conditions. TRAP, a regulator possibly involved in pathogenesis, was expressed only in RF122 while proteins from the Isd system, involved in heme acquisition, were exclusive to Sau302 and Sau340. In summary, the metabolic differences suggest a reduced virulence of the strains causing subclinical mastitis which may contribute to the persistent infection seen in the animals.

Downregulated RPL6 inhibits lung cancer cell proliferation and migration and promotes cell apoptosis by regulating the AKT signaling pathway.

BackgroundLung cancer is still one of the most common causes of cancer-related mortality, and the overall survival is less than 5%. It is important and necessary to elucidate the mechanism of lung cancer progression. Recently, more and more research has demonstrated that many ribosomal proteins (RPs) are dysregulated in tumors. Among these RPs, ribosomal protein L6 (RPL6) is reported to promote cell growth and cell cycle progression in gastric cancer cells through upregulating cyclin E. However, its function in lung cancer is still unknown.MethodsWe first explored RPL6 expression in lung cancer samples. Next, we used gene knockdown to analyze the unknown regulatory role of RPL6 in lung cancer carcinoma and lung cancer cell lines. Furthermore, we explored the potential signaling pathway of RPL6 with Western blotting.ResultsIn this study, we demonstrated that RPL6 expression was highly expressed in lung cancer tissues and lung cancer cell lines. RPL6 downregulation inhibited H1299 and H2975 cell proliferation, migration and induced cell apoptosis. Also RPL6 downregulation increased the protein levels of cleaved caspase-3 and Bax, while decreasing the protein level of B-cell lymphoma 2 (Bcl-2). Western blotting results showed that proteins activating the AKT signaling pathway, such as p-AKT and p-S6, were downregulated in RPL6 knockdown lung cancer cells.ConclusionsThese findings show that RPL6 can be used as a potential therapeutic and preventive biomarker in lung cancer treatment and prognosis by regulating the AKT signaling pathway.

Identification and detection of mutations potentially associated with decreased susceptibility to macrolides and lincomycin in Mycoplasma anserisalpingitidis isolates

Mycoplasma anserisalpingitidis infection is associated with the inflammation of the genital tract and cloaca, embryo lethality and decreased egg production in geese, leading to serious economic losses. This bacterium has so far been described in Europe and Asia. There is no commercially available vaccine against M. anserisalpingitidis, thus treatment of waterfowl mycoplasmosis relies mainly on antimicrobial therapy. However, M. anserisalpingitidis isolates with decreased susceptibility to macrolides and lincomycin have been reported before.The minimal inhibitory concentration (MIC) values of tilmicosin, tylosin, tylvalosin and lincomycin were determined against 82 M. anserisalpingitidis isolates originating from Hungary, Poland, China and Vietnam. Whole-genome sequence analyses revealed two mutations in the 23S rRNA coding regions and one mutation in the 50S ribosomal protein L22 coding gene possibly correlating with decreased susceptibility to the examined antibiotics. Mismatch amplification mutation assays coupled with melt analysis (melt-MAMAs) were designed to detect the nucleotide substitutions.This study is the first to describe resistance-related mutations in the goose pathogen M. anserisalpingitidis. The developed molecular assays support targeted antibiotic usage, hence their use may help to reduce the development and spread of antibiotic resistance.

Identification of Key Genes and Pathways Involved in Circulating Tumor Cells in Colorectal Cancer.

Background Characterization of the features associated with circulating tumor cells (CTCs) is one of major interests for predicting clinical outcome of colorectal cancer (CRC) patients. However, the molecular features of CTCs remain largely unclear. Methods For identification of key genes and pathways, GSE31023, contained CTCs from six metastatic CRC patients and three controls, was retrieved for differentially expressed gene (DEG) analysis. Protein-protein interaction networks of DEGs were constructed. Hub genes from the network were prognostic analyzed, as well as the association with tumor-infiltrating immune cells. Results 1353 DEGs were identified between the CTC and control groups, with 403 genes upregulated and 950 downregulated. 32 pathways were significantly enriched in KEGG, with ribosome pathway as top. The top 10 hub genes were included, including eukaryotic translation elongation factor 2 (EEF2), ribosomal protein S2 (RPS2), ribosomal protein S5 (RPS5), ribosomal protein L3 (RPL3), ribosomal protein S3 (RPS3), ribosomal protein S14 (RPS14), ribosomal protein SA (RPSA), eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), ribosomal protein S15a (RPS15A), and ribosomal protein L4 (RPL4). The correlation between CD4+ T cells and RPS14 (correlation = −0.5) was the highest in colon cancer while CD8+ T and RPS2 (correlation = −0.53) was the highest in rectal cancer. Conclusion This study identified potential role of ribosome pathway in CTC, providing further insightful therapeutic targets and biomarkers for CRC.

Loss of RPS27a expression regulates the cell cycle, apoptosis, and proliferation via the RPL11-MDM2-p53 pathway in lung adenocarcinoma cells

BackgroundDepletion of certain ribosomal proteins induces p53 activation, which is mediated mainly by ribosomal protein L5 (RPL5) and/or ribosomal protein L11 (RPL11). Therefore, RPL5 and RPL11 may link RPs and p53 activation. Thus, this study aimed to explore whether RPs interact with RPL11 and regulate p53 activation in lung adenocarcinoma (LUAD) cells.MethodsThe endogenous RPL11-binding proteins in A549 cells were pulled down through immunoprecipitation and identified with a proteomics approach. Docking analysis and GST-fusion protein assays were used to analyze the interaction of ribosomal protein S27a (RPS27a) and RPL11. Co-immunoprecipitation and in vitro ubiquitination assays were used to detect the effects of knockdown of RPS27a on the interaction between RPS27a and RPL11, and on p53 accumulation. Cell cycle, apoptosis, cell invasion and migration, cell viability and colony-formation assays were performed in the presence of knockdown of RPS27a. The RPS27a mRNA expression in LUAD was analyzed on the basis of the TCGA dataset, and RPS27a expression was detected through immunohistochemistry in LUAD samples. Finally, RPS27a and p53 expression was analyzed through immunohistochemistry in A549 cell xenografts with knockdown of RPS27a.ResultsRPS27a was identified as a novel RPL11 binding protein. GST pull-down assays revealed that RPS27a directly bound RPL11. Knockdown of RPS27a weakened the interaction between RPS27a and RPL11, but enhanced the binding of RPL11 and murine double minute 2 (MDM2), thereby inhibiting the ubiquitination and degradation of p53 by MDM2. Knockdown of RPS27a stabilized p53 in an RPL11-dependent manner and induced cell viability inhibition, cell cycle arrest and apoptosis in a p53-dependent manner in A549 cells. The expression of RPS27a was upregulated in LUAD and correlated with LUAD progression and poorer prognosis. Overexpression of RPS27a correlated with upregulation of p53, MDM2 and RPL11 in LUAD clinical specimens. Knockdown of RPS27a increased p53 activation, thus, suppressing the formation of A549 cell xenografts in nude mice.ConclusionsRPS27a interacts with RPL11, and RPS27a knockdown enhanced the binding of RPL11 and MDM2, thereby inhibiting MDM2-mediated p53 ubiquitination and degradation; in addition, RPS27a as important roles in LUAD progression and prognosis, and may be a therapeutic target for patients with LUAD.Graphical

Identification of ribosomal protein L9 as a novel regulator of proinflammatory damage-associated molecular pattern molecules.

We previously reported that advanced glycation endproducts (AGEs) increase the proinflammatory activity of high mobility group box-1 (HMGB1), a representative damage-associated molecular pattern molecule (DAMP), through their direct interaction. This suggested that AGEs activate other DAMPs and led us to search for novel DAMPs capable of interacting with AGEs. The chromatographic analysis using AGE-immobilized gel revealed the ribosomal protein family to be a factor with binding activity to AGEs. Ribosomal protein L9 (RPL9), a member of the ribosomal protein family, was found in the centrifugal supernatant of ruptured cells and in the serum of lipopolysaccharide (LPS)-stimulated sepsis model mice, exhibiting similar characteristic properties to HMGB1. Although HMGB1 potentiated LPS-stimulated TNF-α expression in macrophage-like RAW264.7 cells, RPL9 hardly exhibited this activity. Of note, RPL9 significantly suppressed the potentiated mRNA expression and protein production of TNF-α by HMGB1 plus LPS stimulation, suggesting its regulatory roles in DAMP-induced proinflammatory activity. Based on the differential scanning fluorimetric analysis, the direct interaction between RPL9 and HMGB1 may play a role in the suppressive effects of RPL9. This study suggested that RPL9 is a novel type of DAMP with a regulatory role in the proinflammatory response and provided insight into the pathophysiology of inflammatory diseases.

The Identification and Validation of Hub Genes Associated with Acute Myocardial Infarction Using Weighted Gene Co-Expression Network Analysis.

Acute myocardial infarction (AMI), one of the most severe and fatal cardiovascular diseases, remains the main cause of mortality and morbidity worldwide. The objective of this study is to investigate the potential biomarkers for AMI based on bioinformatics analysis. A total of 2102 differentially expressed genes (DEGs) were screened out from the data obtained from the gene expression omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) explored the co-expression network of DEGs and determined the key module. The brown module was selected as the key one correlated with AMI. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses demonstrated that genes in the brown module were mainly enriched in ‘ribosomal subunit’ and ‘Ribosome’. Gene Set Enrichment Analysis revealed that ‘TNFA_SIGNALING_VIA_NFKB’ was remarkably enriched in AMI. Based on the protein–protein interaction network, ribosomal protein L9 (RPL9) and ribosomal protein L26 (RPL26) were identified as the hub genes. Additionally, the polymerase chain reaction (PCR) results indicated that the expression levels of RPL9 and RPL26 were both downregulated in AMI patients compared with controls, in accordance with the bioinformatics analysis. In summary, the identified DEGs, modules, pathways, and hub genes provide clues and shed light on the potential molecular mechanisms of AMI.

Reproductive tissue-specific translatome of a rice thermo-sensitive genic male sterile line

Translational regulation, especially tissue- or cell type-specific gene regulation, plays essential roles in plant growth and development. Thermo-sensitive genic male sterile (TGMS) lines have been widely used for hybrid breeding in rice (Oryza sativa). However, little is known about translational regulation during reproductive stage in TGMS rice. Here, we use translating ribosome affinity purification (TRAP) combined with RNA sequencing to investigate the reproductive tissue-specific translatome of TGMS rice expressing FLAG-tagged ribosomal protein L18 (RPL18) from the germline-specific promoter MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1). Differentially expressed genes at the transcriptional and translational levels are enriched in pollen and anther-related formation and development processes. These contain a number of genes reported to be involved in tapetum programmed cell death (PCD) and lipid metabolism during pollen development and anther dehiscence in rice, including several encoding transcription factors and key enzymes, as well as several long non-coding RNAs (lncRNAs) that potentially affect tapetum and pollen-related genes in male sterility. This study represents the comprehensive reproductive tissue-specific characterization of the translatome in TGMS rice. These results contribute to our understanding of the molecular basis of sterility in TGMS rice and will facilitate further genetic manipulation of TGMS rice in two-line breeding systems.

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells.

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10‐R98S). Our data support that RPL10‐R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock‐in of RPL10‐R98S was associated with a 1.7‐ to 2.5‐fold increased production of four transiently expressed recombinant proteins and 1.7‐fold for one out of two stably expressed proteins. In CHO‐S cells, eGFP reached a 2‐fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10‐R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10‐R98S phenotypes can maximize its exploitability for the mammalian protein production industry.

Identification of ribosomal protein L24 (RPL24) from the oriental river prawn, Macrobrachium nipponense, and its roles in ovarian development

Ribosomal proteins exhibit various extraribosomal functions in addition to their roles in protein synthesis. In this study, complementary DNA (cDNA) of ribosomal protein L24 in Macrobrachium nipponense (MnRPL24) was isolated, and its role in ovarian development was investigated using quantitative real-time PCR (qPCR), immunohistochemistry (IHC), RNA interference (RNAi) and histological observations. The complete cDNA of MnRPL24 is 564 base pairs (bps) and contains a 486 bp open reading frame (ORF) encoding 162 amino acids (aas). The highest expression level of MnRPL24 among eight tissues was found in the ovary, specifically in the stage I ovary. The MnRPL24 protein existed in the cytoplasm and nucleus of developing oocytes, and also existed in the cytoplasm of follicle cells in developing ovaries. After MnRPL24 knockdown by RNAi, the expression levels of vitellogenin (Vg), vitellogenin receptor (Vgr), cyclin-dependent kinase 2 (Cdc2) and M-phase cyclin (Cyclin B) genes and the gonadsomatic index (GSI) did not show the typical trend of gradually elevation with ovarian development and finally decrease in the later stage of ovarian cycle. Moreover, the oviposition rate (OR) was downregulated, and oocyte development was delayed after MnRPL24 knockdown. After eyestalk ablation, the MnRPL24 expression level was considerably elevated in the initial stages and decreased in the late stage of the ovarian development cycle. This investigation illustrates a possible regulatory role of MnRPL24 in the ovarian development of M. nipponense, and MnRPL24 may act as a stimulator of early ovarian development.

Selection and Validation of Reference Genes for RT-qPCR Normalization in Bradysia odoriphaga (Diptera: Sciaridae) Under Insecticides Stress.

Bradysia odoriphaga (Diptera: Sciaridae) is the most serious root maggot pest which causes substantial damage to the Chinese chive. Organophosphate (OP) and neonicotinoid insecticides are widely used chemical pesticides and play important roles in controlling B. odoriphaga. However, a strong selection pressure following repeated pesticide applications has led to the development of resistant populations of this insect. To understand the insecticide resistance mechanism in B. odoriphaga, gene expression analysis might be required. Appropriate reference gene selection is a critical prerequisite for gene expression studies, as the expression stability of reference genes can be affected by experimental conditions, resulting in biased or erroneous results. The present study shows the expression profile of nine commonly used reference genes [elongation factor 1α (EF-1α), actin2 (ACT), elongation factor 2α (EF-2α), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), ubiquitin-conjugating enzyme (UBC), and α-tubulin (TUB)] was systematically analyzed under insecticide stress. Moreover, we also evaluated their expression stability in other experimental conditions, including developmental stages, sexes, and tissues. Five programs (NormFinder, geNorm, BestKeeper, RefFinder, and ΔCt) were used to validate the suitability of candidate reference genes. The results revealed that the most appropriate sets of reference genes were RPL10 and ACT across phoxim; ACT and TUB across chlorpyrifos and chlorfluazuron; EF1α and TUB across imidacloprid; EF1α and EF2α across developmental stages; RPL10 and TUB across larvae; EF1α and ACT across tissues, and ACT and G6PDH across sex. These results will facilitate the standardization of RT-qPCR and contribute to further research on B. odoriphaga gene function under insecticides stress.

Evaluation of qPCR reference genes for taimen (Hucho taimen) under heat stress

As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen.

RplI interacts with 5' UTR of exsA to repress its translation and type III secretion system in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen capable of causing variety of infections in humans. The type III secretion system (T3SS) is a critical virulence determinant of P. aeruginosa in the host infections. Expression of the T3SS is regulated by ExsA, a master regulator that activates the expression of all known T3SS genes. Expression of the exsA gene is controlled at both transcriptional and posttranscriptional levels. Here, we screened a P. aeruginosa transposon (Tn5) insertional mutant library and found rplI, a gene coding for the ribosomal large subunit protein L9, to be a repressor for the T3SS gene expression. Combining real-time quantitative PCR (qPCR), western blotting and lacZ fusion assays, we show that RplI controls the expression of exsA at the posttranscriptional level. Further genetic experiments demonstrated that RplI mediated control of the exsA translation involves 5’ untranslated region (5’ UTR). A ribosome immunoprecipitation assay and qPCR revealed higher amounts of a 24 nt fragment from exsA mRNA being associated with ribosomes in the ΔrplI mutant. An interaction between RplI and exsA mRNA harboring its 24 nt, but not 12 nt, 5’ UTR was confirmed by RNA Gel Mobility Shift and Microscale Thermophoresis assays. Overall, this study identifies the ribosomal large subunit protein L9 as a novel T3SS repressor that inhibits ExsA translation in P. aeruginosa.

RPL17 Promotes Colorectal Cancer Proliferation and Stemness through ERK and NEK2/β-catenin Signaling Pathways.

Aims: Ribosomal protein L17 (RPL17), a 60S subunit component, is up-regulated in colorectal cancer (CRC). However, its oncogenic role in CRC progression remains unexplored. Thus, we aimed to investigate the effect of RPL17 targeting on CRC in vitro and in vivo and whether RPL17 gained an extra-ribosomal function during CRC development.Methods: RPL17-specific siRNAs complexed with cationic lipids were transfected to CRC cells to silence target gene expression and then real-time RT-PCR and western blotting were applied to observe the change of expression or activity of genes or proteins of interest. Cell proliferation assay, clonogenic assay and cell cycle analysis were used to determine the in vitro effects of RPL17siRNAs on CRC cell growth, and a subcutaneous xenograft assay was applied to test the effect of RPL17siRNAs on in vivo tumor growth. RNA sequencing and western blotting were used to investigate the underlying mechanisms. Sphere-forming assay, invasion assay and migration assay were used to evaluate the effects of RPL17siRNAs on CRC stemness.Results: siRNA-mediated inhibition of RPL17 expression suppressed CRC cell growth and long-term colony formation by inducing apoptotic cell death. Similarly, targeting RPL17 effectively suppressed tumor formation in a mouse xenograft model. RNA sequencing of RPL17-silenced CRC cells revealed the same directional regulation of 159 (93 down- and 66 up-regulated) genes. Notably, NIMA-related kinase 2 (NEK2), which functionally cooperates with extracellular-regulated protein kinase (ERK) and plays a pivotal role in mitotic progression and stemness maintenance, was down-regulated. RPL17 silencing reduced NEK2, β-catenin, and p-ERK protein levels. These molecular alterations reflected the reduction in sphere-forming capacity, expression of stem cell marker genes, migration, and invasion. Reversely, RPL17 overexpression increased the ability of long-term colony formation, migration, and invasion.Conclusion: Our findings indicate that RPL17 promotes CRC proliferation and stemness via the ERK and NEK2/β-catenin signaling axis, and targeting RPL17 could be the next molecular strategy for both primary CRC treatment and prevention of secondary tumor formation.

Prediction of Angiopoietin-like Protein 4-related Signaling Pathways in Cholangiocarcinoma Cells.

Angiopoietin-like protein 4 (ANGPTL4) is a multifunctional signaling protein implicated in carbohydrate metabolism, inflammation, cancer growth and progression, anoikis resistance, angiogenesis, and metastasis. However, signaling pathways of ANGPTL4 in cholangiocarcinoma (CCA) remain unknown. The aim of this study was to explore ANGPTL4-related signaling proteins and pathways associated with CCA biology. ANGPTL4 of CCA cells was silenced by small interfering RNA (siRNA) with scramble control and ANGPTL4-related signaling proteins were investigated using mass spectrometry, bioinformatics tools and molecular docking. Among the 321 differentially expressed proteins, 151 were down-regulated. Among them, bioinformatic analyses revealed that ANGPTL4 interacts with DNA-dependent protein kinase catalytic subunit (PRKDC) and 60S ribosomal protein L21 (RPL21) via AKT serine/threonine kinase 1 (AKT1), mechanistic target of rapamycin kinase (MTOR) and ribosomal protein L5 (RPL5). Online database analysis showed that mRNA and protein expression levels of ANGPTL4-related signaling proteins were significantly higher in CCA than in normal tissues. Moreover, a high mRNA expression level was associated with high tumor grade (p<0.0001) and lymph node metastasis (p<0.0001). The signaling pathway of ANGPTL4 in CCA progression might be regulated by PRKDC and RPL21. Furthermore, high expression of ANGPTL4-related signaling proteins has potential to be used in clinical prognosis.

Regulation of inflammatory response based on interaction among AGEs, DAMPs, and/or cytokines

Advanced Glycation End Products (AGEs) are produced through a non-enzymatic reaction between reducing sugar and biomolecules. These molecules are suggested to stimulate several receptors and activate inflammatory reactions. Because the accumulation of AGEs was found to be associated with hyperglycemia and/or aging, these molecules should be contributed to the pathogenesis of inflammatory diseases related to these conditions. Interestingly, possible receptors to engage AGEs are common to endogenous proinflammatory factors called damage-associated molecular pattern molecules (DAMPs). This raised the possibility that the action mechanism of AGEs and DAMPs is closely correlated. Previously, we found that AGEs interacted with high mobility group box-1 (HMGB1), a representative DAMP, and that this interaction activated the proinflammatory activity of HMGB1. These findings suggested that exacerbation of inflammation induced by HMGB1 was caused by the condition accumulating AGEs. In addition, AGEs were found to change the action of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) through their direct interaction. TWEAK is called a multifunctional cytokine, and is suggested to regulate tumor necrosis factor-α (TNF-α) induced inflammatory reactions. We found that coexistence of AGEs and TWEAK inhibited this action of TWEAK, suggesting that accumulation of AGEs induces exacerbation of inflammation induced by TNF-α. Furthermore, we found ribosomal protein L9 (RPL9) as a novel AGE-binding protein. RPL9 inhibited HMGB1-induced inflammatory reaction, suggesting that RPL9 is the endogenous regulator for DAMPs. These findings suggested that there is a novel mechanism to regulate inflammatory reactions through the interaction among AGEs, DAMPs, and/or cytokines.

Ribosomal Protein Database Profiling Lends Clarity to Ribosomal Protein Evolution and Mass Distribution

Existence of theoretical ribosomal protein mass fingerprint as well as utility of ribosomal protein as biomarkers in mass spectrometry microbial identification suggests phylogenetic significance for this class of proteins. To serve the above two functions, facile means of identifying and extracting important attributes of ribosomal proteins from proteome data file of microbial species must be found. Additionally, there is a need to calculate important properties of ribosomal proteins such as molecular weight and nucleotide sequence based on amino acid sequence information from FASTA proteome file. This work sought to support the above endeavour through developing MATLAB software that extracts the amino acid sequence information of all ribosomal proteins from the FASTA proteome data file of a microbial species downloaded from UniProt. Built-in functions in MATLAB are subsequently employed to calculate important properties of extracted ribosomal proteins such as number of amino acid residue, molecular weight and nucleotide sequence. All information above are output, as a database, to an Excel file for ease of storage and retrieval. Data available from the analysis of an Escherichia coli K-12 proteome revealed that the bacterium possesses a total of 59 ribosomal proteins distributed between the large and small ribosome subunits. The ribosomal protein ranges in sequence length from 38 (50S ribosomal protein L36) to 557 (30S ribosomal protein S1). In terms of molecular weight distribution, the profiled ribosomal proteins range in weight from 4364.305 Da (50S ribosomal protein L36) to 61157.66 Da (30S ribosomal protein S1). More important, analysis of the distribution of the molecular weight of different ribosomal proteins in E. coli reveals a smooth curve that suggests strong co-evolution of ribosomal protein sequence and mass given the tight constraints that a functional ribosome presents. Finally, cluster analysis reveals a preponderance of small ribosomal proteins compared to larger ones, which remains to be a mystery to evolutionary biologists. Overall, the information encapsulated in the ribosomal protein database should find use in gaining a better appreciation for the molecular weight distribution of ribosomal proteins in a species, as well as delivering information for using ribosomal protein biomarkers in identifying particular microbial species in mass spectrometry microbial identification.