Regulation Of Ribosomal Proteins Research Articles

Multiplexing of TMT labeling reveals folate-deficient diet-specific proteome changes in NTDs.

Introduction: In the early stage of embryonic development, the neural tube (NT) cannot be closed properly due to some complex factors, including environmental factors, genetic factors, and the relationship between various factors, leading to the occurrence of neural tube defects (NTDs). Methods: In this study, we induced a mouse model of NTDs by feeding mice with a low-folate diet and intraperitoneally injecting them with 1.5mg/kg methotrexate on E7.5. Fetal mice were achieved at E13.5, and we extracted proteins from brain tissues with trypsin digestion. After enzymatic digestion, peptides were labeled with TMT/iTRAQ and separated in high-performance liquid chromatography (HPLC) for subsequent liquid chromatography tandem mass spectroscopy (LC-MS/MS) analysis. We used gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation to analyze proteomic changes and analyze the functional enrichment of differentially expressed proteins (DEPs) in the NTD mice tissues. Results: A low-folate-induced mouse model was successfully constructed. Folate was used as a sensitizing agent, and the teratogenicity rate of the NTD fetal mice increased to 36.5% when the concentration of methotrexate was at 1.5mg/kg. Mass spectrometry was used to identify 6,614 proteins, and among them, 5,656 proteins were quantified. In the following proteomic analysis, GO classification and KEGG pathway enrichment analysis were conducted, and heatmaps were drawn for differentially expressed proteins (DEPs). The main pathways associated with NTDs, such as the Hedgehog, Wnt, p53, and Hippo signaling pathways and the one-carbon pool mediated by folate, can be identified through a protein-protein interaction (PPI) network. It was also found that the regulation of ribosomal proteins, such as RPL13 and RPL14, which are upregulated in NTDs, has a certain impact on neural tube development. Discussion: Our results revealed proteomic changes in the tissues of low-folate-induced NTD mice. Validation showed that ribosomal proteins play a regulatory role during the development of NTDs and provides new ideas for the pathogenesis and preventive measures of NTDs.

Liver proteomic analysis of Bai-Hu-Tang treatment to systemic inflammatory response

IntroductionBai-Hu-Tang (BHT), a classical prescription of traditional Chinese medicine, has been extensively used to treat many infectious diseases and high fever syndrome. However, the specific mechanisms and molecular targets affected by BHT have not been thoroughly investigated in the context of treating systemic inflammatory response. This study aimed to explore the comprehensive protein mechanism of BHT during treatment of the lipopolysaccharide (LPS) systemic inflammatory response. MethodsThe rabbit model of the systemic inflammatory response was established by LPS intravenous injection (15 μg/kg·bw), and aqueous extract of BHT was administrated by gavage to the model rabbits. At the same time, a group of normal rabbits were also gavaged with BHT alone. Based on liver histopathological study, total liver protein was extracted, and two-dimensional liquid chromatography (LC) - tandem mass spectrometry (MS) coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling analysis was employed to identify and quantify the liver proteome. Differentially expressed proteins (DEPs) were screened through comparing with control animals, and bioinformatics analysis was conducted to explore kyoto encyclopedia of genes and genomes (KEGG) pathways enrichment and protein clustering among different groups. Real time polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of 4 ribosomal proteins to verify the proteomic results. ResultsThe results showed that ribosome biogenesis is the most significant biological process and ribosomal DEPs mainly distribute in the large subunit of ribosome during the BHT treatment to the LPS systemic inflammatory response. RT-PCR confirmed that BHT had an effect on mRNA expression of ribosome proteins. DiscussionTherefore, the biological pathway of ribosome biogenesis and the regulation of ribosomal proteins may be the cruicial factors when employing BHT for the treatment of the systemic inflammatory response induced by LPS.

GSK3α Regulates Temporally Dynamic Changes in Ribosomal Proteins upon Amino Acid Starvation in Cancer Cells.

Amino acid availability is crucial for cancer cells' survivability. Leukemia and colorectal cancer cells have been shown to resist asparagine depletion by utilizing GSK3-dependent proteasomal degradation, termed the Wnt-dependent stabilization of proteins (Wnt/STOP), to replenish their amino acid pool. The inhibition of GSK3α halts the sourcing of amino acids, which subsequently leads to cancer cell vulnerability toward asparaginase therapy. However, resistance toward GSK3α-mediated protein breakdown can occur, whose underlying mechanism is poorly understood. Here, we set out to define the mechanisms driving dependence toward this degradation machinery upon asparagine starvation in cancer cells. We show the independence of known stress response pathways including the integrated stress response mediated with GCN2. Additionally, we demonstrate the independence of changes in cell cycle progression and expression levels of the asparagine-synthesizing enzyme ASNS. Instead, RNA sequencing revealed that GSK3α inhibition and asparagine starvation leads to the temporally dynamic downregulation of distinct ribosomal proteins, which have been shown to display anti-proliferative functions. Using a CRISPR/Cas9 viability screen, we demonstrate that the downregulation of these specific ribosomal proteins can rescue cell death upon GSK3α inhibition and asparagine starvation. Thus, our findings suggest the vital role of the previously unrecognized regulation of ribosomal proteins in bridging GSK3α activity and tolerance of asparagine starvation.

Homeostatic regulation of ribosomal proteins by ubiquitin-independent cotranslational degradation

Ribosomes are the workplace for protein biosynthesis. Protein production required for normal cell function is tightly linked to ribosome abundance. It is well known that ribosomal genes are actively transcribed and ribosomal messenger RNAs (mRNAs) are rapidly translated, and yet ribosomal proteins have relatively long half-lives. These observations raise questions as to how homeostasis of ribosomal proteins is controlled. Here, we show that ribosomal proteins, while posttranslationally stable, are subject to high-level cotranslational protein degradation (CTPD) except for those synthesized as ubiquitin (Ub) fusion precursors. The N-terminal Ub moiety protects fused ribosomal proteins from CTPD. We further demonstrate that cotranslational folding efficiency and expression level are two critical factors determining CTPD of ribosomal proteins. Different from canonical posttranslational degradation, we found that CTPD of all the ribosomal proteins tested in this study does not require prior ubiquitylation. This work provides insights into the regulation of ribosomal protein homeostasis and furthers our understanding of the mechanism and biological significance of CTPD.

Regulation of Ribosomal Protein Synthesis in Mycobacteria: The Autogenous Control of rpsO.

The autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. In this work, taking the rpsO gene as a classic example, we addressed for the first time the in vivo regulation of r-protein synthesis in the mycobacteria M. smegmatis (Msm) and M. tuberculosis (Mtb). We used a strategy based on chromosomally integrated reporters under the control of the rpsO regulatory regions and the ectopic expression of Msm S15 to measure its impact on the reporter expression. Because the use of E. coli as a host appeared inefficient, a fluorescent reporter system was developed by inserting Msm or Mtb rpsO-egfp fusions into the Msm chromosome and expressing Msm S15 or E. coli S15 in trans from a novel replicative shuttle vector, pAMYC. The results of the eGFP expression measurements in Msm cells provided evidence that the rpsO gene in Msm and Mtb was feedback-regulated at the translation level. The mutagenic analysis showed that the folding of Msm rpsO 5′UTR in a pseudoknot appeared crucial for repression by both Msm S15 and E. coli S15, thus indicating a striking resemblance of the rpsO feedback control in mycobacteria and in E. coli.

Fermented Feed Modulates Meat Quality and Promotes the Growth of Longissimus Thoracis of Late-Finishing Pigs.

Simple SummaryLittle is known about the effect of fermented feed on lean mass production of finishing pigs and the underlying mechanism. This study aimed to investigate the effect of fermented diet on growth performance, carcass traits, meat quality and growth of longissimus thoracis (LT) of finishing pigs. Our results found that fermented diet significantly increased loin eye area and lean mass percentage, decreased backfat thickness and improved meat quality of LT by decreasing shear force and drip loss at 48 h post slaughter and improving meat sensory characteristics compared with control diet. Fermented diet also increased the abundance of insulin, insulin receptor, myoblast determination protein (MyoD) and myosin heavy chain-I (MyHC-I) transcripts, and the phosphorylation levels of protein kinase B (AKT), mammalian target of rapamycin (mTORC1), initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase 1 (S6K1) in LT, while decreasing the expression of muscle atrophy F-box (MAFbx) and forkhead Box O1 (Foxo1) mRNA transcripts. Our results indicated that fermented diet improved meat quality and enhanced LT growth of finishing pigs by increasing insulin/AKT/mTORC1 protein synthesis cascade and activating Foxo1/MAFbx pathway, along with the regulation of ribosomal protein and proteins involved in muscle contraction and muscle hypertrophy. This study investigated the effect of fermented diet on growth performance, carcass traits, meat quality and growth of longissimus thoracis (LT) of finishing pigs. A total of 48 finishing pigs [Duroc × (Landrace × Large White), male, 126 ± 5-d-old] weighing 98.76 ± 1.27 kg were randomly assigned to two treatments (eight pens per treatment and three pigs per pen) for a 28-d feeding trial, including control diet and fermented diet. Fermented diet significantly increased the loin eye area and lean mass percentage, decreased backfat thickness and improved meat quality of LT by decreasing the shear force and drip loss at 48 h post slaughter and improving meat sensory characteristics compared with control diet. A fermented diet also significantly increased the abundance of insulin, insulin receptor (IR), myoblast determination protein (MyoD) and myosin heavy chain-I (MyHC-I) transcripts, and the phosphorylation levels of AKT, mTORC1, 4EBP1 and S6K1 in LT, while decreasing the expression of muscle atrophy F-box (MAFbx) and forkhead Box O1 (Foxo1) mRNA transcripts. Moreover, proteomic analysis revealed that differentially expressed proteins predominantly involved in protein synthesis and muscle development were modulated by fermented diet. Our results indicated that a fermented diet improved meat quality and enhanced LT growth of finishing pigs by increasing insulin/AKT/mTORC1 protein synthesis cascade and activating the Foxo1/MAFbx pathway, along with the regulation of ribosomal protein and proteins involved in muscle contraction and muscle hypertrophy.

Selective Translation of Low Abundance and Upregulated Transcripts in Halobacterium salinarum.

When organisms encounter an unfavorable environment, they transition to a physiologically distinct, quiescent state wherein abundant transcripts from the previous active growth state continue to persist, albeit their active transcription is downregulated. In order to generate proteins for the new quiescent physiological state, we hypothesized that the translation machinery must selectively translate upregulated transcripts in an intracellular milieu crowded with considerably higher abundance transcripts from the previous active growth state. Here, we have analyzed genome-wide changes in the transcriptome (RNA sequencing [RNA-seq]), changes in translational regulation and efficiency by ribosome profiling across all transcripts (ribosome profiling [Ribo-seq]), and protein level changes in assembled ribosomal proteins (sequential window acquisition of all theoretical mass spectra [SWATH-MS]) to investigate the interplay of transcriptional and translational regulation in Halobacterium salinarum as it transitions from active growth to quiescence. We have discovered that interplay of regulatory processes at different levels of information processing generates condition-specific ribosomal complexes to translate preferentially pools of low abundance and upregulated transcripts. Through analysis of the gene regulatory network architecture of H. salinarum, Escherichia coli, and Saccharomyces cerevisiae, we demonstrate that this conditional, modular organization of regulatory programs governing translational systems is a generalized feature across all domains of life.IMPORTANCE Our findings demonstrate conclusively that low abundance and upregulated transcripts are preferentially translated, potentially by environment-specific translation systems with distinct ribosomal protein composition. We show that a complex interplay of transcriptional and posttranscriptional regulation underlies the conditional and modular regulatory programs that generate ribosomes of distinct protein composition. The modular regulation of ribosomal proteins with other transcription, translation, and metabolic genes is generalizable to bacterial and eukaryotic microbes. These findings are relevant to how microorganisms adapt to unfavorable environments when they transition from active growth to quiescence by generating proteins from upregulated transcripts that are in considerably lower abundance relative to transcripts associated with the previous physiological state. Selective translation of transcripts by distinct ribosomes could form the basis for adaptive evolution to new environments through a modular regulation of the translational systems.

Regulation of Ribosomal Proteins on Viral Infection.

Ribosomal proteins (RPs), in conjunction with rRNA, are major components of ribosomes involved in the cellular process of protein biosynthesis, known as “translation”. The viruses, as the small infectious pathogens with limited genomes, must recruit a variety of host factors to survive and propagate, including RPs. At present, more and more information is available on the functional relationship between RPs and virus infection. This review focuses on advancements in my own understanding of critical roles of RPs in the life cycle of viruses. Various RPs interact with viral mRNA and proteins to participate in viral protein biosynthesis and regulate the replication and infection of virus in host cells. Most interactions are essential for viral translation and replication, which promote viral infection and accumulation, whereas the minority represents the defense signaling of host cells by activating immune pathway against virus. RPs provide a new platform for antiviral therapy development, however, at present, antiviral therapeutics with RPs involving in virus infection as targets is limited, and exploring antiviral strategy based on RPs will be the guides for further study.

Protein Profiling of Arabidopsis Roots Treated With Humic Substances: Insights Into the Metabolic and Interactome Networks.

Background and Aim: Humic substances (HSs) influence the chemical and physical properties of the soil, and are also known to affect plant physiology and nutrient uptake. This study aimed to elucidate plant metabolic pathways and physiological processes influenced by HS activity.Methods: Arabidopsis roots were treated with HS for 8 h. Quantitative mass spectrometry-based proteomics analysis of root proteins was performed using the iTRAQ (Isobaric Tag for Relative and Absolute Quantification) technique. Out of 902 protein families identified and quantified for HS treated vs. untreated roots, 92 proteins had different relative content. Bioinformatic tools such as STRING, KEGG, IIS and Cytoscape were used to interpret the biological function, pathway analysis and visualization of network amongst the identified proteins.Results: From this analysis it was possible to evaluate that all of the identified proteins were functionally classified into several categories, mainly redox homeostasis, response to inorganic substances, energy metabolism, protein synthesis, cell trafficking, and division.Conclusion: In the present study an overview of the metabolic pathways most modified by HS biological activity is provided. Activation of enzymes of the glycolytic pathway and up regulation of ribosomal protein indicated a stimulation in energy metabolism and protein synthesis. Regulation of the enzymes involved in redox homeostasis suggest a pivotal role of reactive oxygen species in the signaling and modulation of HS-induced responses.

Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translation regulation of mRNAs encoding ribosomal proteins

The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it is unclear whether mechanistic target of rapamycin (mTOR)-4EBP1/2 is the exclusive translation regulator of this group of genes, and furthermore, systematic searches for novel translation modulators have been immensely challenging because of difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for translation modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translation effects in a manner that is dependent on GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a translation assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a noncanonical, mTOR-independent mode for translation regulation of ribosomal proteins.

CBNSimulator: a simulator tool for understanding the behaviour of complex biomolecular networks using discrete time simulation

Because of the lack of satisfactory solutions to explain biological systems, biologists usually focus on modelling and simulation tools to understand the behaviour of these complex organisms. Indeed, computational modelling and simulation of cells plays a pivotal role in systems biology. In this paper, we tackle the problem of studying the behaviour of human cells by reproducing the behaviour of complex biomolecular networks. To this end, we present in this paper an approach for simulating complex biomolecular networks inspired by the discrete-event simulation model (DEVS), a formalism developed for supporting the modelling of complex systems. In this paper, we propose a simulation tool, named ”CBNSimulator”, based on a logical model of the biomolecular network and taking advantage of the performance of a discrete-time simulation model for understanding the evolution and the behaviour of complex biomolecular networks as a discrete sequence of events in time. The proposed tool has been applied to the case study of a ribosomal protein regulation network, named ”the bacteriophage T4 gene 32”, and results given by this simulation tool are in agreement with the expert’s judgement. Moreover, the graphical user interface of CBNSimulator allows biologists to easily reproduce, analyse and understand behaviour of complex biomolecular networks through discrete simulation.

Role of CK2-dependent phosphorylation of Ifh1 and Crf1 in transcriptional regulation of ribosomal protein genes in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Fhl1 is involved in the regulation of ribosomal protein (RP) genes through interaction with either its coactivator Ifh1 or corepressor Crf1, depending on nutrient conditions. Interaction of Fhl1 with Ifh1 or Crf1 is achieved through a forkhead-associated (FHA) domain of Fhl1, which binds to forkhead-binding (FHB) domains of Ifh1 and Crf1. Here, we demonstrate that CK2-dependent phosphorylation of T681 and T348 residues, located in the FHB domains of Ifh1 and Crf1, respectively, provides binding sites for the FHA domain of Fhl1. Cells expressing Ifh1T681A mutant showed reduced association of Ifh1 at the RP gene promoters and decreased levels of RP gene transcripts, thereby reducing the growth rate. On the other hand, cells expressing Crf1T348A showed a defect in repressing RP gene transcription upon inhibition of target of rapamycin complex 1 (TORC1) by rapamycin treatment. Taken together, these findings suggest the mechanisms by which CK2-dependent recruitment of Ifh1 and Crf1 at the RP gene promoters governs the transcription of RP genes.

GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies

The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies.

Identification, molecular characterization and expression analysis of RPL24 genes in three Cucurbitaceae family members: cucumber, melon and watermelon

Ribosomal proteins are crucial for the proper growth and development of any organism, including plants. The ribosomal protein L24 (RPL24) is found in the large subunit of the ribosome and is responsible for the stabilization of the peptidyl transferase activity. Although RPL24 gene has been individually identified in different organisms, little is known about the genome-wide survey and expression patterns of R24 genes in Cucurbitaceae family members. We identified seven Cucurbitaceae RPL24 genes from cucumber, melon and watermelon. They were phylogenetically clustered into seven major groups. Gene structure and motif composition are relatively conserved in each group. Three-dimensional homology modelling of RPL24 proteins was performed with higher confidence level. CmRPL24-01 was isolated from melon and characterized at a molecular level. The regulation of ribosomal proteins in melon under drought stress conditions was also studied. The expression of CmRPL24-01 gene increased in melon leaf tissue at 3 h upon polyethylene glycol treatment and showed a gradual induction after 12 h. Our study provided a very useful reference for identification and functional analysis of RPL24 protein members in different plants. In addition, this research indicated a potential usage of ribosomal proteins in response to drought stress.

Transcriptional Downregulation of Rice rpL32 Gene under Abiotic Stress Is Associated with Removal of Transcription Factors within the Promoter Region

BackgroundThe regulation of ribosomal proteins in plants under stress conditions has not been well studied. Although a few reports have shown stress-specific post-transcriptional and translational mechanisms involved in downregulation of ribosomal proteins yet stress-responsive transcriptional regulation of ribosomal proteins is largely unknown in plants.Methodology/Principal FindingsIn the present work, transcriptional regulation of genes encoding rice 60S ribosomal protein L32 (rpL32) in response to salt stress has been studied. Northern and RT-PCR analyses showed a significant downregulation of rpL32 transcripts under abiotic stress conditions in rice. Of the four rpL32 genes in rice genome, the gene on chromosome 8 (rpL32_8.1) showed a higher degree of stress-responsive downregulation in salt sensitive rice variety than in tolerant one and its expression reverted to its original level upon withdrawal of stress. The nuclear run-on and promoter:reporter assays revealed that the downregulation of this gene is transcriptional and originates within the promoter region. Using in vivo footprinting and electrophoretic mobility shift assay (EMSA), cis-elements in the promoter of rpL32_8.1 showing reduced binding to proteins in shoots of salt stressed rice seedlings were identified.ConclusionsThe present work is one of the few reports on study of stress downregulated genes. The data revealed that rpL32 gene is transcriptionally downregulated under abiotic stress in rice and that this transcriptional downregulation is associated with the removal of transcription factors from specific promoter elements.

Compensation for differences in gene copy number among yeast ribosomal proteins is encoded within their promoters.

Coordinate regulation of ribosomal protein (RP) genes is key for controlling cell growth. In yeast, it is unclear how this regulation achieves the required equimolar amounts of the different RP components, given that some RP genes exist in duplicate copies, while others have only one copy. Here, we tested whether the solution to this challenge is partly encoded within the DNA sequence of the RP promoters, by fusing 110 different RP promoters to a fluorescent gene reporter, allowing us to robustly detect differences in their promoter activities that are as small as ~10%. We found that single-copy RP promoters have significantly higher activities, suggesting that proper RP stoichiometry is indeed partly encoded within the RP promoters. Notably, we also partially uncovered how this regulation is encoded by finding that RP promoters with higher activity have more nucleosome-disfavoring sequences and characteristic spatial organizations of these sequences and of binding sites for key RP regulators. Mutations in these elements result in a significant decrease of RP promoter activity. Thus, our results suggest that intrinsic (DNA-dependent) nucleosome organization may be a key mechanism by which genomes encode biologically meaningful promoter activities. Our approach can readily be applied to uncover how transcriptional programs of other promoters are encoded.

Expression Profiling and Regulation of Genes Related to Silkworm Posterior Silk Gland Development and Fibroin Synthesis

The posterior silk gland (PSG) is the most important suborgan responsible for the synthesis and secretion of silk core fibroin proteins in silkworm. Here, we performed genome-scale expression profiling analysis of silkworm PSG at the fourth molting (M4) and at day 1 (V1), day 3 (V3), day 5 (V5), and wandering stage (W) of the fifth instar by microarray analysis with 22 987 probes. We found that the five genes of silk proteins secreted from PSG including fibroin heavy (H) and light (L) chains, P25, seroin 1, and seroin 2 basically showed obvious up-regulation at V3 which lasted to V5, while slight down-regulation at W. The expression of translation-related genes including ribosomal proteins and translation initiation factors generally remained stable from M4 to V5, whereas it showed clear down-regulation at W. Clustering analysis of the 643 significantly differentially expressed transcripts revealed that 43 of the important genes including seroin 1 and sugar transporter protein had co-expression patterns which were consistent with the rate changes of fibroin synthesis and PSG growth. Pathway analysis disclosed that the genes in different clusters might have co-regulations and direct interactions. These genes were supposed to be involved in the fibroin synthesis and secretion. The differential expression of several hormone-related genes also suggested their functions on the regulation of PSG development and fibroin synthesis. 2D gel-based proteomics and phosphoproteomics profiling revealed that the phosphorylated proteins accounted for no more than one-sixth of the total proteins at each stage, which was much lower than the level in normal eukaryotic cells. Changes in the phosphorylation status and levels of several proteins such as actin-depolymerizing factor 1 and enolase might be deeply involved in fibroin secretion and tissue development. Shotgun proteomic profiling combined with label-free quantification analysis on the PSG at V3, V5, and W revealed that many small heat shock proteins (sHSP) were specially expressed at W, which was substantially consistent with the results from 2-DE analysis, and implied the close correlations of sHSP with the physiological states of PSG at W. A majority of significantly up-regulated proteins at V5 were related to ribosome pathway, which was different from the microarray results, implying that the translation-level regulation of ribosomal proteins might be critical for fibroin synthesis. In contrast, the ubiquitin-proteasome pathway related proteins appeared obviously up-regulated at W, suggesting that the programmed cell death process of PSG cells might be started before cocooning.

Proteasomal degradation of Sfp1 contributes to the repression of ribosome biogenesis during starvation and is mediated by the proteasome activator Blm10

The regulation of ribosomal protein (RP) gene transcription is tightly linked to the nutrient status of the cell and is under the control of metabolic signaling pathways. In Saccharomyces cerevisiae several transcriptional activators mediate efficient RP gene transcription during logarithmic growth and dissociate from RP gene promoters upon nutrient limitation. Repression of RP gene transcription appears to be regulated predominantly by posttranslational modification and cellular localization of transcriptional activators. We report here that one of these factors, Sfp1, is degraded by the proteasome and that the proteasome activator Blm10 is required for regulated Sfp1 degradation. Loss of Blm10 results in the stabilization and increased nuclear abundance of Sfp1 during nutrient limitation, increased transcription of RP genes, increased levels of RPs, and decreased rapamycin-induced repression of RP genes. Thus we conclude that proteasomal degradation of Sfp1 is mediated by Blm10 and contributes to the repression of ribosome biogenesis under nutrient depletion.

Perturbation of 60 S Ribosomal Biogenesis Results in Ribosomal Protein L5- and L11-dependent p53 Activation

Ribosomal proteins play an important role in p53 activation in response to nucleolar stress. Multiple ribosomal proteins, including L5, L11, L23, and S7, have been shown to bind to and inhibit MDM2, leading to p53 activation. However, it is not clear whether ribosomal protein regulation of MDM2 is specific to some, but not all ribosomal proteins. Here we show that L29 and L30, two ribosomal proteins from the 60 S ribosomal subunit, do not bind to MDM2 and do not inhibit MDM2-mediated p53 suppression, indicating that the ribosomal protein regulation of the MDM2-p53 feedback loop is specific. Interestingly, direct perturbation of the 60 S ribosomal biogenesis by knocking down either L29 or L30 drastically induced the level and activity of p53, leading to p53-depedent cell cycle arrest. This p53 activation was drastically inhibited by knockdown of L11 or L5. Consistently, knockdown of L29 or L30 enhanced the interaction of MDM2 with L11 and L5 and markedly inhibited MDM2-mediated p53 ubiquitination, suggesting that direct perturbation of 60 S ribosomal biogenesis activates p53 via L11- and L5-mediated MDM2 suppression. Mechanistically, knockdown of L30 or L29 significantly increased the NEDDylation and nuclear retention of L11. Knocking down endogenous NEDD8 suppressed p53 activation induced by knockdown of L30. These results demonstrate that NEDDylation of L11 plays a critical role in mediating p53 activation in response to perturbation of ribosomal biogenesis.

Quantitation of the Ribosomal Protein Autoregulatory Network Using Mass Spectrometry

Relative levels of ribosomal proteins were quantified in crude cell lysates using mass spectrometry. A method for quantifying cellular protein levels using macromolecular standards is presented that does not require complex sample separation, identification of high-responding peptides, affinity purification, or postgrowth modifications. Perturbations in ribosomal protein levels by overexpression of individual proteins correlate to known autoregulatory mechanisms and extend the network of ribosomal protein regulation.