BAIAP2L1 accelerates breast cancer progression and chemoresistance by activating AKT signaling through binding with ribosomal protein L3.

BAI1-associated protein 2-like 1 (BAIAP2L1), also known as insulin receptor tyrosine kinase substrate, modulates the insulin network; however, its function in breast cancer has not been explored. Immunohistochemical analysis of 140 breast cancer specimens (77 triple-negative and 63 nontriple-negative cases) indicated that BAIAP2L1 expression was higher in breast cancer tissues (56/140, 40%) than in normal breast tissues (28.3%, 15/53; p < 0.001). BAIAP2L1 expression in breast cancer was correlated with triple-negative breast cancer (p = 0.0013), advanced TNM stage (p = 0.001), lymph node metastasis (p = 0.001), and poor patient prognosis (p = 0.001). BAIAP2L1 overexpression could accelerate breast cancer proliferation, invasion, and stemness in vivo and in vitro, possibly through the activation of AKT, Snail, and cyclin D1. Treatment with the AKT inhibitor LY294002 reduced the effects of BAIAP2L1 overexpression on breast cancer cells. BAIAP2L1 may bind to the AA202-288 of ribosomal protein L3 (RPL3) within its SRC homology 3 (SH3) domain, the loss of which may abolish the transduction of the AKT signaling pathway by promoting the degradation of PIK3CA. Moreover, BAIAP2L1 overexpression may induce chemotherapy resistance, with BAIAP2L1 expression being higher in patients with advanced Miller grades than those with lower grades. Our results indicated that BAIAP2L1 promotes breast cancer progression through the AKT signaling pathway by interacting with RPL3 through its SH3 domain.

METTL18-mediated histidine methylation of RPL3 modulates translation elongation for proteostasis maintenance.

Protein methylation occurs predominantly on lysine and arginine residues, but histidine also serves as a methylation substrate. However, a limited number of enzymes responsible for this modification have been reported. Moreover, the biological role of histidine methylation has remained poorly understood to date. Here, we report that human METTL18 is a histidine methyltransferase for the ribosomal protein RPL3 and that the modification specifically slows ribosome traversal on Tyr codons, allowing the proper folding of synthesized proteins. By performing an in vitro methylation assay with a methyl donor analog and quantitative mass spectrometry, we found that His245 of RPL3 is methylated at the τ-N position by METTL18. Structural comparison of the modified and unmodified ribosomes showed stoichiometric modification and suggested a role in translation reactions. Indeed, genome-wide ribosome profiling and an in vitro translation assay revealed that translation elongation at Tyr codons was suppressed by RPL3 methylation. Because the slower elongation provides enough time for nascent protein folding, RPL3 methylation protects cells from the cellular aggregation of Tyr-rich proteins. Our results reveal histidine methylation as an example of a ribosome modification that ensures proteome integrity in cells.

Suppression of RPL34 Inhibits Tumor Cell Proliferation and Promotes Apoptosis in Glioblastoma.

Accumulating evidence indicates Ribosomal protein 34 (RPL34) promotes tumor malignance and its expression is associated with poor prognosis in multiple cancer cells. However, the physiological role and biological mechanism of RPL34 in glioblastoma (GBM) remain unclear. Hence, this study aimed to investigate the expression and the role of RPL34 in GBM. A total of 59 glioma samples and 12 normal brains for epilepsy surgery were used to determine the underlying mechanisms and the biological behaviors of RPL34 in GBM. In this study, we identified that RPL34 expression was significantly (p < 0.05) enriched in GBM tumors compared with low-grade glioma and normal brain, and its expression was associated with poor survival. Additionally, RPL34 was functionally required for tumor proliferation in vitro. Mechanically, inhibition of RPL34 induced glioma cell apoptosis by activation of Bad/Caspase7/PARP signaling pathway. The RPL34 promotes cell survival in GBM and could be a potential therapeutic target for GBM.

The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly.

Early pre-60S ribosomal particles are poorly characterized, highly dynamic complexes that undergo extensive rRNA folding and compaction concomitant with assembly of ribosomal proteins and exchange of assembly factors. Pre-60S particles contain numerous RNA helicases, which are likely regulators of accurate and efficient formation of appropriate rRNA structures. Here we reveal binding of the RNA helicase Dbp7 to domain V/VI of early pre-60S particles in yeast and show that in the absence of this protein, dissociation of the Npa1 scaffolding complex, release of the snR190 folding chaperone, recruitment of the A3 cluster factors and binding of the ribosomal protein uL3 are impaired. uL3 is critical for formation of the peptidyltransferase center (PTC) and is responsible for stabilizing interactions between the 5' and 3' ends of the 25S, an essential pre-requisite for subsequent pre-60S maturation events. Highlighting the importance of pre-ribosome remodeling by Dbp7, our data suggest that in the absence of Dbp7 or its catalytic activity, early pre-ribosomal particles are targeted for degradation.

Phylogeography of Baryancistrus xanthellus (Siluriformes: Loricariidae), a rheophilic catfish endemic to the Xingu River basin in eastern Amazonia.

Baryancistrus xanthellus (Loricariidae) is an endemic fish species from the Xingu River basin with its life history in the shallow rapid waters flowing over bedrock substrates. In order to investigate the genetic diversity and demographic history of B. xanthellus we analyzed sequence data for one mitochondrial gene (Cyt b) and introns 1 and 5 of nuclear genes Prolactin (Prl) and Ribosomal Protein L3 (RPL3). The analyses contain 358 specimens of B. xanthellus from 39 localities distributed throughout its range. The number of genetically diverged groups was estimated using Bayesian inference on Cyt b haplotypes. Haplotype networks, AMOVA and pairwise fixation index was used to evaluate population structure and gene flow. Historical demography was inferred through neutrality tests and the Extended Bayesian Skyline Plot (EBSP) method. Five longitudinally distributed Cyt b haplogroups for B. xanthellus were identified in the Xingu River and its major tributaries, the Bacajá and Iriri. The demographic analysis suggests that rapids habitats have expanded in the Iriri and Lower Xingu rivers since 200 ka (thousand years) ago. This expansion is possibly related to an increase in water discharge as a consequence of higher rainfall across eastern Amazonia. Conversely, this climate shift also would have promoted zones of sediment trapping and reduction of rocky habitats in the Xingu River channel upstream of the Iriri River mouth. Populations of B. xanthellus showed strong genetic structure along the free-flowing river channels of the Xingu and its major tributaries, the Bacajá and Iriri. The recent impoundment of the Middle Xingu channel for the Belo Monte hydroelectric dam may isolate populations at the downstream limit of the species distribution. Therefore, future conservation plans must consider the genetic diversity of B. xanthellus throughout its range.

Screening of viral-vectored P. falciparum pre-erythrocytic candidate vaccine antigens using chimeric rodent parasites.

To screen for additional vaccine candidate antigens of Plasmodium pre-erythrocytic stages, fourteen P. falciparum proteins were selected based on expression in sporozoites or their role in establishment of hepatocyte infection. For preclinical evaluation of immunogenicity of these proteins in mice, chimeric P. berghei sporozoites were created that express the P. falciparum proteins in sporozoites as an additional copy gene under control of the uis4 gene promoter. All fourteen chimeric parasites produced sporozoites but sporozoites of eight lines failed to establish a liver infection, indicating a negative impact of these P. falciparum proteins on sporozoite infectivity. Immunogenicity of the other six proteins (SPELD, ETRAMP10.3, SIAP2, SPATR, HT, RPL3) was analyzed by immunization of inbred BALB/c and outbred CD-1 mice with viral-vectored (ChAd63 or ChAdOx1, MVA) vaccines, followed by challenge with chimeric sporozoites. Protective immunogenicity was determined by analyzing parasite liver load and prepatent period of blood stage infection after challenge. Of the six proteins only SPELD immunized mice showed partial protection. We discuss both the low protective immunogenicity of these proteins in the chimeric rodent malaria challenge model and the negative effect on P. berghei sporozoite infectivity of several P. falciparum proteins expressed in the chimeric sporozoites.

Knockdown of Muscle-Specific Ribosomal Protein L3-Like Enhances Muscle Function in Healthy and Dystrophic Mice.

Ribosomal protein L3-like (RPL3L) is a poorly characterized ribosomal protein that is exclusively expressed in skeletal and cardiac muscle. RPL3L is also downregulated in Duchenne muscular dystrophy (DMD), suggesting that it may play an important role in muscle biology. In this study, we investigated the role of RPL3L in skeletal muscle of healthy C57 and dystrophic mdx mice. We show that RPL3L is developmentally regulated and that intramuscular adeno-associated virus (AAV)-mediated RPL3L knockdown in the tibialis anterior of C57 and mdx mice results in increased specific force with improved resistance to eccentric contraction induced muscle damage in dystrophic muscles. The mechanism by which RPL3L knockdown improves muscle function remains unclear. Histological observations showed a significant increase in muscle length and decrease in muscle cross-sectional area after RPL3L inhibition suggesting that this ribosomal protein may play a role in myofiber morphology. The endogenous downregulation of RPL3L in DMD may be a protective mechanism that attempts to improve skeletal muscle function and counteract the dystrophic phenotype.

Comprehensive analysis of molecular pathways and key genes involved in lumbar disc herniation.

ABSTRACT: Based on the Thompson classification of intervertebral discs (IVDs), we systematically analyzed gene expression differences between severely degenerated and mildly degenerated IVDs and explored the underlying molecular mechanisms using bioinformatics methods and multichip integration. We used multiomics analysis, includes mRNA microarray and methylation chips, to explore the genetic network and mechanisms of lumbar disc herniation (LDH). Subsequently, the Combat function of the R language SVA package was applied to eliminate heterogeneity between the gene expression data. And the protein-protein interaction (PPI) network, gene ontology (GO), and molecular pathways were used to constructs the mechanisms network. Consequently, we obtained 149 differentially expressed genes. Related molecular pathways are the following: ribosome activity, oxidative phosphorylation, extracellular matrix response. Besides, through PPI network analysis, genes with higher connectivity such as UBA52, RPLP0, RPL3, RPLP2, and RPL27 were also identified, suggesting that they play important regulatory roles in the complex network associated with LDH. Additionally, cg12556991 (RPL27) and cg06852319 (RPLP0) were found to be LDH-related candidate DNA methylation modification sites in the IVDs tissue of LDH patients. In conclusions, ribosome activity, oxidative phosphorylation, and extracellular matrix response may be potential molecular mechanisms underlying LDH, while hub genes involved in UBA52, RPLP0, RPL3, RPLP2, and RPL27, and candidate DNA methylation modification sites of cg12556991and cg06852319 are likely key regulators in the development of LDH.

Human METTL18 is a histidine-specific methyltransferase that targets RPL3 and affects ribosome biogenesis and function.

Protein methylation occurs primarily on lysine and arginine, but also on some other residues, such as histidine. METTL18 is the last uncharacterized member of a group of human methyltransferases (MTases) that mainly exert lysine methylation, and here we set out to elucidate its function. We found METTL18 to be a nuclear protein that contains a functional nuclear localization signal and accumulates in nucleoli. Recombinant METTL18 methylated a single protein in nuclear extracts and in isolated ribosomes from METTL18 knockout (KO) cells, identified as 60S ribosomal protein L3 (RPL3). We also performed an RPL3 interactomics screen and identified METTL18 as the most significantly enriched MTase. We found that His-245 in RPL3 carries a 3-methylhistidine (3MH; τ-methylhistidine) modification, which was absent in METTL18 KO cells. In addition, both recombinant and endogenous METTL18 were found to be automethylated at His-154, thus further corroborating METTL18 as a histidine-specific MTase. Finally, METTL18 KO cells displayed altered pre-rRNA processing, decreased polysome formation and codon-specific changes in mRNA translation, indicating that METTL18-mediated methylation of RPL3 is important for optimal ribosome biogenesis and function. In conclusion, we have here established METTL18 as the second human histidine-specific protein MTase, and demonstrated its functional relevance.

Geographic information system-based analysis of COVID-19 cases in India during pre-lockdown, lockdown, and unlock phases.

OBJECTIVE: The World Health Organization formally announced the global COVID-19 pandemic on March 11, 2020 due to widespread infections. In this study, COVID-19 cases in India were critically analyzed during the pre-lockdown (PLD), lockdown (LD), and unlock (UL) phases. METHOD: Analyses were conducted using geospatial technology at district, state, and country levels, and comparisons were also made with other countries throughout the world that had the highest infection rates. India had the third highest infection rate in the world after the USA and Brazil during UL2.0-UL3.0 phases, the second highest after the USA during UL4.0-UL5.0 phases, and the highest among South Asian Association for Regional Cooperation (SAARC) countries in PLD-UL5.0 period. RESULTS: The trend in the number of COVID-19 cases was associated with the population density where higher numbers tended to be record in the eastern, southern, and west-central parts of India. The death rate in India throughout the pandemic period under study was lower than the global average. Kerala reported the maximum number of infections during PLD whereas Maharashtra had the highest numbers during all LD and UL phases. Eighty percent of the cases in India were concentrated mainly in highly populous districts. CONCLUSION: The top 25 districts accounted for 70.99%, 69.38%, 54.87%, 44.23%, 40.48%, and 38.96% of the infections from the start of UL1.0 until the end of UL phases, respectively, and the top 26-50 districts accounted for 6.38%, 6.76%, 11.23%, 12.98%, 13.40%, and 13.61% of cases in these phase, thereby indicating that COVID-19 cases spread during the UL period. By October 31, 2020, Delhi had the highest number of infections, followed by Bengaluru Urban, Pune, Mumbai, Thane, and Chennai. No decline in the infection rate occurred, even in UL5.0, thereby indicating a highly alarming situation in India.

Mechanisms of Linezolid Resistance Among Clinical Staphylococcus spp. in Spain: Spread of Methicillin- and Linezolid-Resistant S. epidermidis ST2.

This study aimed at determining the mechanisms of linezolid resistance and the molecular characteristics of clinical Staphylococcus aureus (n = 2) and coagulase-negative staphylococci (n = 15) isolates obtained from four Spanish hospitals. The detection of linezolid resistance mechanisms (mutations and acquisition of resistance genes) was performed by PCR/sequencing. The antimicrobial resistance and virulence profile was determined, and the isolates were typed by different molecular techniques. Moreover, the genetic environment of the cfr gene was determined by whole-genome sequencing. The cfr gene was detected in one methicillin-resistant S. aureus (MRSA) that also displayed the amino acid change Val118Ala in the ribosomal protein L4. The second S. aureus isolate was methicillin susceptible and showed different alterations in the ribosomal protein L4. All remaining linezolid-resistant Staphylococcus epidermidis (n = 14) and Staphylococcus hominis isolates (n = 1) showed the mutation G2576T (n = 14) or C2534T (n = 1) in the 23S rRNA. Moreover, different amino acid changes were detected in the ribosomal proteins L3 and L4 in S. epidermidis isolates. All S. epidermidis isolates belonged to the multilocus sequence type ST2. Linezolid-resistant staphylococci (LRS) showed a multiresistance phenotype, including methicillin resistance that was detected in all isolates but one, and was mediated by the mecA gene. The cfr gene in the MRSA isolate was located together with the fexA gene on a conjugative 38,864 bp plasmid. Linezolid- and methicillin-resistant S. epidermidis ST2 showing mutations in the 23S rRNA and in the ribosomal proteins L3 and L4 are spread among Spanish hospitals, whereas LRS carrying acquired linezolid resistance genes are rarely detected.

DUOX2 promotes the progression of colorectal cancer cells by regulating the AKT pathway and interacting with RPL3.

Dual oxidase 2 (DUOX2) is an important regulatory protein in the organic process of thyroid hormone iodine. Mounting evidence suggests that DUOX2 plays a crucial role in the occurrence and development of cancers. However, the function and mechanism of DUOX2 in colorectal cancer (CRC) have not been fully clarified. In the present study, the relationship between the expression of DUOX2 and the clinicopathological features and prognosis of CRC patients was analyzed. Furthermore, the effects of DUOX2 on proliferation and invasion in vitro and in vivo were examined. DUOX2-associated proteins were identified by immunoprecipitation (IP). Next-generation sequencing detection was performed to illustrate the mechanism of DUOX2 in CRC cells. It was found that the expression levels of DUOX2 in metastatic sites were significantly higher than those in primary tumor tissues, and this was demonstrated to be associated with poor prognosis. The knockdown of DUOX2 inhibited the invasion and migration of CRC cells. Furthermore, DUOX2 regulated the stability of ribosomal protein uL3 (RPL3) by affecting the ubiquitination status of RPL3, and the invasion and migration ability of DUOX2 can be reversed by the overexpression of RPL3. The downregulation of DUOX2 can affect the expression level of a large number of genes, and a number of these are enriched in the PI3K-AKT pathway. Some of the changes caused by DUOX2 can be reversed by RPL3. In summary, DUOX2 exhibits a significantly higher expression in CRC tumor samples, and facilitates the invasion and metastasis ability of CRC cells by interacting with RPL3.

S-Adenosyl-l-Methionine Overcomes uL3-Mediated Drug Resistance in p53 Deleted Colon Cancer Cells.

PURPOSE: In order to study novel therapeutic approaches taking advantage of natural compounds showing anticancer and anti-proliferative effects, we focused our interest on S-adenosyl-l-methionine, a naturally occurring sulfur-containing nucleoside synthesized from adenosine triphosphate and methionine by methionine adenosyltransferase, and its potential in overcoming drug resistance in colon cancer cells devoid of p53. RESULTS: In the present study, we demonstrated that S-adenosyl-l-methionine overcomes uL3-mediated drug resistance in p53 deleted colon cancer cells. In particular, we demonstrated that S-adenosyl-l-methionine causes cell cycle arrest at the S phase; inhibits autophagy; augments reactive oxygen species; and induces apoptosis in these cancer cells. CONCLUSIONS: Results reported in this paper led us to propose S-adenosyl-l-methionine as a potential promising agent for cancer therapy by examining p53 and uL3 profiles in tumors to yield a better clinical outcomes.

Prognostic factor identification by analysis of the gene expression and DNA methylation data in glioma.

Objective: This study was aimed to identify prognostic factors in glioma by analysis of the gene expression and DNA methylation data. Methods: The RNAseq and DNA methylation data associated with glioma were downloaded from GEO and TCGA databases to analyze the differentially expressed genes (DEGs) and methylated genes between tumor and normal tissues. Function and pathway analyses, co-expression network and survival analysis were performed based on these DEGs. The intersection genes of DEGs and differentially methylated genes were obtained followed by function analysis. Results: Total 2190 DEGs were identified between tumor and normal tissues, which were significantly enriched in neuron differentiation associated functions, as well as ribosome pathway. There were 6186 methylation sites (2834 up-regulated and 3352 down-regulated) with significant differences in tumor vs. normal. In the constructed co-expression network, DPP6, MAPK10 and RPL3 were hub genes. Survival analysis of 20 DEGs obtained 18 prognostic genes, among which 9 were differentially methylated, such as LHFPL tetraspan subfamily member 3 (LHFPL3), cadherin 20 (CDH20), complexin 2 (CPLX2), and tenascin R (TNR). The intersection of DEGs and differentially methylated genes (632 genes) were significantly enriched in functions of neuron differentiation. Conclusion: DPP6, MAPK10 and RPL3 may play important roles in tumorigenesis of glioma. Additionally, methylation of LHFPL3, CDH20, CPLX2, and TNR may serve as prognostic factors of glioma.

Bi-allelic missense disease-causing variants in RPL3L associate neonatal dilated cardiomyopathy with muscle-specific ribosome biogenesis.

Dilated cardiomyopathy (DCM) belongs to the most frequent forms of cardiomyopathy mainly characterized by cardiac dilatation and reduced systolic function. Although most cases of DCM are classified as sporadic, 20-30% of cases show a heritable pattern. Familial forms of DCM are genetically heterogeneous, and mutations in several genes have been identified that most commonly play a role in cytoskeleton and sarcomere-associated processes. Still, a large number of familial cases remain unsolved. Here, we report five individuals from three independent families who presented with severe dilated cardiomyopathy during the neonatal period. Using whole-exome sequencing (WES), we identified causative, compound heterozygous missense variants in RPL3L (ribosomal protein L3-like) in all the affected individuals. The identified variants co-segregated with the disease in each of the three families and were absent or very rare in the human population, in line with an autosomal recessive inheritance pattern. They are located within the conserved RPL3 domain of the protein and were classified as deleterious by several in silico prediction software applications. RPL3L is one of the four non-canonical riboprotein genes and it encodes the 60S ribosomal protein L3-like protein that is highly expressed only in cardiac and skeletal muscle. Three-dimensional homology modeling and in silico analysis of the affected residues in RPL3L indicate that the identified changes specifically alter the interaction of RPL3L with the RNA components of the 60S ribosomal subunit and thus destabilize its binding to the 60S subunit. In conclusion, we report that bi-allelic pathogenic variants in RPL3L are causative of an early-onset, severe neonatal form of dilated cardiomyopathy, and we show for the first time that cytoplasmic ribosomal proteins are involved in the pathogenesis of non-syndromic cardiomyopathies.

Emergence of linezolid-resistant Staphylococcus epidermidis in the tertiary children's hospital in Cracow, Poland.

Coagulase-negative staphylococci, ubiquitous commensals of human skin, and mucous membranes represent important pathogens for immunocompromised patients and neonates. The increasing antibiotic resistance among Staphylococcus epidermidis is an emerging problem worldwide. In particular, the linezolid-resistant S. epidermidis (LRSE) strains are observed in Europe since 2014. The aim of our study was to genetically characterize 11 LRSE isolates, recovered mostly from blood in the University Children's Hospital in Krakow, Poland, between 2015 and 2017. For identification of the isolates at the species level, we used 16S rRNA sequencing and RFLP of the saoC gene. Isolates were characterized phenotypically by determining their antimicrobial resistance patterns and using molecular methods such as PFGE, MLST, SCCmec typing, detection of the ica operon, and analysis of antimicrobial resistance determinants. All isolates were multidrug-resistant, including resistance to methicillin, and exhibited so-called PhLOPSA phenotype. In PFGE, all isolates (excluding one from a catheter) represented identical patterns, were identified as ST2, and harbored the ica operon, responsible for biofilm formation. Linezolid resistance was associated with acquisition of A157R mutation in the ribosomal protein L3 and the presence of cfr gene. All isolates revealed new SCCmec cassette element composition. Recently, pediatric patients with serious staphylococcal infections are often treated with linezolid. The increasing linezolid resistance in bacterial strains becomes a real threat for patients, and monitoring such infections combined with surveillance and infection prevention programs is very important to decrease number of linezolid-resistant staphylococcal strains.

uL3 Mediated Nucleolar Stress Pathway as a New Mechanism of Action of Antiproliferative G-quadruplex TBA Derivatives in Colon Cancer Cells.

The antiproliferative G-quadruplex aptamers are a promising and challenging subject in the framework of the anticancer therapeutic oligonucleotides research field. Although several antiproliferative G-quadruplex aptamers have been identified and proven to be effective on different cancer cell lines, their mechanism of action is still unexplored. We have recently described the antiproliferative activity of a heterochiral thrombin binding aptamer (TBA) derivative, namely, LQ1. Here, we investigate the molecular mechanisms of LQ1 activity and the structural and antiproliferative properties of two further TBA derivatives, differing from LQ1 only by the small loop base-compositions. We demonstrate that in p53 deleted colon cancer cells, LQ1 causes nucleolar stress, impairs ribosomal RNA processing, leading to the accumulation of pre-ribosomal RNAs, arrests cells in the G2/M phase and induces early apoptosis. Importantly, the depletion of uL3 abrogates all these effects, indicating that uL3 is a crucial player in the mechanism of action of LQ1. Taken together, our findings identify p53-independent and uL3-dependent nucleolar stress as a novel stress response pathway activated by a specific G-quadruplex TBA derivative. To the best of our knowledge, this investigation reveals, for the first time, the involvement of the nucleolar stress pathway in the mechanism of action of antiproliferative G-quadruplex aptamers.

Role of uL3 in the Crosstalk between Nucleolar Stress and Autophagy in Colon Cancer Cells.

The nucleolus is the site of ribosome biogenesis and has been recently described as important sensor for a variety of cellular stressors. In the last two decades, it has been largely demonstrated that many chemotherapeutics act by inhibiting early or late rRNA processing steps with consequent alteration of ribosome biogenesis and activation of nucleolar stress response. The overall result is cell cycle arrest and/or apoptotic cell death of cancer cells. Our previously data demonstrated that ribosomal protein uL3 is a key sensor of nucleolar stress activated by common chemotherapeutic agents in cancer cells lacking p53. We have also demonstrated that uL3 status is associated to chemoresistance; down-regulation of uL3 makes some chemotherapeutic drugs ineffective. Here, we demonstrate that in colon cancer cells, the uL3 status affects rRNA synthesis and processing with consequent activation of uL3-mediated nucleolar stress pathway. Transcriptome analysis of HCT 116p53-/- cells expressing uL3 and of a cell sub line stably depleted of uL3 treated with Actinomycin D suggests a new extra-ribosomal role of uL3 in the regulation of autophagic process. By using confocal microscopy and Western blotting experiments, we demonstrated that uL3 acts as inhibitory factor of autophagic process; the absence of uL3 is associated to increase of autophagic flux and to chemoresistance. Furthermore, experiments conducted in presence of chloroquine, a known inhibitor of autophagy, indicate a role of uL3 in chloroquine-mediated inhibition of autophagy. On the basis of these results and our previous findings, we hypothesize that the absence of uL3 in cancer cells might inhibit cancer cell response to drug treatment through the activation of cytoprotective autophagy. The restoration of uL3 could enhance the activity of many drugs thanks to its pro-apoptotic and anti-autophagic activity.

Protein Histidine Methylation.

Protein histidine methylation is a rarely studied posttranslational modification in eukaryotes. Although the presence of N-methylhistidine was demonstrated in actin in the early 1960s, so far, only a limited number of proteins containing N-methylhistidine have been reported, including S100A9, myosin, skeletal muscle myosin light chain kinase (MLCK 2), and ribosomal protein Rpl3. Furthermore, the role of histidine methylation in the functioning of the protein and in cell physiology remains unclear due to a shortage of studies focusing on this topic. However, the molecular identification of the first two distinct histidine-specific protein methyltransferases has been established in yeast (Hpm1) and in metazoan species (actin-histidine N-methyltransferase), giving new insights into the phenomenon of protein methylation at histidine sites. As a result, we are now beginning to recognize protein histidine methylation as an important regulatory mechanism of protein functioning whose loss may have deleterious consequences in both cells and in organisms. In this review, we aim to summarize the recent advances in the understanding of the chemical, enzymological, and physiological aspects of protein histidine methylation.

Ribosomal protein uL3 targets E2F1 and Cyclin D1 in cancer cell response to nucleolar stress.

Several experimental strategies in the treatment of cancer include drug alteration of cell cycle regulatory pathways as a useful strategy. Extra-ribosomal functions of human ribosomal protein L3 (uL3) may affect DNA repair, cell cycle arrest and apoptosis. In the present study, we demonstrated that uL3 is required for the activation of G1/S transition genes. Luciferase assays established that uL3 negatively regulates the activity of E2F1 promoter. Induced ribosome-free uL3 reduces Cyclin D1 mRNA and protein levels. Using protein/protein immunoprecipitation methods, we demonstrated that uL3 physically interacts with PARP-1 affecting E2F1 transcriptional activity. Our findings led to the identification of a new pathway mediated by uL3 involving E2F1 and Cyclin D1 in the regulation of cell cycle progression.