Large Subunit Binding Research Articles

Meta-Analysis of EGF-Stimulated Normal and Cancer Cell Lines to Discover EGF-Associated Oncogenic Signaling Pathways and Prognostic Biomarkers.

Although epidermal growth factor (EGF) controls many crucial processes in the human body, it can increase the risk of developing cancer when overexpresses. This study focused on detecting cancer-associated genes that are dysregulated by EGF overexpression. To identify differentially expressed genes (DEGs), two independent meta-analyses with normal and cancer RNA-Seq samples treated by EGF were conducted. The new DEGs detected only via two meta-analyses were used in all downstream analyses. To reach count data, the tools of FastQC, Trimmomatic, HISAT2, SAMtools, and HTSeq-count were employed. DEGs in each individual RNA-Seq study and the meta-analysis of RNA-Seq studies were identified using DESeq2 and metaSeq R package, respectively. MCODE detected densely interconnected top clusters in the protein-protein interaction (PPI) network of DEGs obtained from normal and cancer datasets. The DEGs were then introduced to Enrichr and ClueGO/CluePedia, and terms, pathways, and hub genes enriched in Gene Ontology (GO) and KEGG and Reactome were detected. The meta-analysis of normal and cancer datasets revealed 990 and 541 new DEGs, all upregulated. A number of DEGs were enriched in protein K48-linked deubiquitination, ncRNA processing, ribosomal large subunit binding, and protein processing in endoplasmic reticulum. Hub genes overexpression (DHX33, INTS8, NMD3, OTUD4, P4HB, RPS3A, SEC13, SKP1, USP34, USP9X, and YOD1) in tumor samples were validated by TCGA and GTEx databases. Overall survival and disease-free survival analysis also confirmed worse survival in patients with hub genes overexpression. The detected hub genes could be used as cancer biomarkers when EGF overexpresses.

Mitoribosomal small subunit maturation involves formation of initiation-like complexes.

Mitochondrial ribosomes (mitoribosomes) play a central role in synthesizing mitochondrial inner membrane proteins responsible for oxidative phosphorylation. Although mitoribosomes from different organisms exhibit considerable structural variations, recent insights into mitoribosome assembly suggest that mitoribosome maturation follows common principles and involves a number of conserved assembly factors. To investigate the steps involved in the assembly of the mitoribosomal small subunit (mt-SSU) we determined the cryoelectron microscopy structures of middle and late assembly intermediates of the Trypanosoma brucei mitochondrial small subunit (mt-SSU) at 3.6- and 3.7-Å resolution, respectively. We identified five additional assembly factors that together with the mitochondrial initiation factor 2 (mt-IF-2) specifically interact with functionally important regions of the rRNA, including the decoding center, thereby preventing premature mRNA or large subunit binding. Structural comparison of assembly intermediates with mature mt-SSU combined with RNAi experiments suggests a noncanonical role of mt-IF-2 and a stepwise assembly process, where modular exchange of ribosomal proteins and assembly factors together with mt-IF-2 ensure proper 9S rRNA folding and protein maturation during the final steps of assembly.

Comparative Analysis of Salinity-Induced Proteomic Changes in Cotton (Gossypium hirsutum L.)

Salt stress on cotton varieties of distinct salinity tolerance can induce expression of different proteins. Zhong 07, a salt-tolerant variety and Zhong s9612, a salt-sensitive variety, were utilized as experimental materials. The leaves of trefoil seedlings treated with or without 0.4% NaCl for 24 h were harvested for whole-protein extraction. Two-dimensional technology, combined with mass spectroscopy (MS) analysis and protein database searching, was employed to detect differentially expressed proteins and determine their identities and biological functions. Compared with the control, Zhong 07 showed 10 differentially expressed proteins under salt stress, of which 6 were upregulated and 4 were downregulated. Meanwhile, 12 differentially expressed proteins were detected in Zhong s9612 under salt stress, of which 10 were upregulated and 2 were downregulated. In the matrix-assisted laser desorption-ionization/time of flight-time of flight/MS analysis, 14 differentially expressed proteins were successfully identified, including the ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisco) large subunit-binding protein subunit alpha (RuBisco α), luminal binding protein (LBP), heat shock protein 70 (Hsp1, 2, 3), pathogenesis-related protein class 10 (PR-10), quinoneoxidoreductase-like protein (QOR), S-adenosylmethioninesyn-thetase (SAMS), enolase (EN), and RuBisco large subunit-binding protein subunit beta (RuBisco β). Cellular function is ultimately executed by proteins, and cotton varieties with different salt tolerance can be influenced by salt stress to various degrees, which can provide certain theoretical foundation for the identification of salt tolerance of cotton varieties. The findings also provide some proteins, such as the RuBisco large subunit binding proteins α and β subunits, OEE2 protein, HSP70, and S-adenosylmethionine synthetase, which can be used as protein markers of salt-to-lerance before- and post-treatment, making a big difference in salt-tolerance identification in cotton.

Proteomics Uncovers a Role for Enhanced Ultraviolet-B Radiation on Wheat Leaves

Wheat (Triticum aestivum), as a kind of important economic crop cultured in the Northern China, is affected by present-day enhanced ultraviolet-B (UV-B) radiation. To get the information of the impact by UV-B radiation on it, the proteins of wheat (Jin mai NO.8) leaves, which were divided into the normal light group (CK) and UV-B radiation group (B), were extracted and ran at SDS-PAGE at different treatment days (5, 6, 7). The proteins were also analyzed by run two-dimensional gel electrophoresis (2-DE), which allowed the identification of some significantly different gel spots. The proteins spots were further verified by Matrix-Assisted Laser Desorption/lonization-time of Flight Mass Spectrometry. The results showed: 1) the enhanced UV-B affects the growth of the wheat, as the visual changes appear on the sixth day; 2) the proteins expressions between the B group and the CK group were remarkably different on the sixth day; 3) the proteins of wheat leaves of the sixth day were further analyzed by 2-DE revealed that twenty-one protein points were identificated between the B group and the CK group. Among these twenty-one proteins, six proteins of them were up-regulated and twelve proteins of them were down-regulated, three new proteins were expressed only in the B group. Three proteins among six proteins, which were up-regulated, were further verified as RuBisCo large subunit binding protein; SOD; Calmodulin. The result indicates wheat could improve genes encoding proteins in their leaves and protect themselves, when enhanced UV-B affects the growth of the wheat.

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO

Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coli. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of in vitro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis.

Ribulose bisphosphate carboxylase assembly: what is the role of the large subunit binding protein?

Abstract Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), is a key enzyme in both photosynthetic carbon fixation and photorespiration. Genetic manipulation of this enzyme to increase its carboxylase activity could lead to substantial increases in net photosynthesis in several important crop plants. Unfortunately, the higher plant enzyme cannot be expressed in active form in E. coli. Several factors may account for this, the best studied of which is a binding protein that interacts with large subunits after they are synthesized but before they are assembled into holoenzyme. Various functions for this binding protein are considered.

Stability and Dissociation of the Large Subunit RuBisCO Binding Protein Complex in Vitro and in Organello

We are studying the stability of the binding protein which associates with newly synthesized large subunits of ribulose bisphosphate carboxylase. In chloroplast extracts, it has been shown that a dodecameric complex of the large subunit binding protein dissociates extensively into binding protein monomers and 7S (117 kilodaltons) large subunit-containing complexes in the presence of ATP. The concentrations of ATP which bring this about are quite low, prompting some investigators to suggest that the dodecameric complex might not exist in vivo. We have found, however, that in concentrated chloroplast extracts, at protein concentrations which are closer to those which occur in organello, the dissociation of the binding protein complex by ATP is much less extensive. For this reason, we have tested the stability of the binding protein in organello, by illuminating chloroplasts followed by lysis and polyacrylamide gel electrophoresis of the extracts. Radioactive large subunits associated with the dodecameric binding protein dissociated extensively in the light. The results are consistent with the idea that the high molecular weight form of the binding protein can function as a reservoir of large subunits which can be tapped in vivo, in a reaction dependent on light and ATP.

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5' non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.

The Rubisco large subunit binding protein

Newly synthesized Rubisco large subunits made by isolated intact chloroplasts fromPisum sativumare bound non-covalently to another protein, termed the Rubisco large subunit binding protein. This protein is implicated in the assembly of Rubisco in higher plant chloroplasts. The binding protein has been purified fromPisum sativumin the form of an oligomer of relative molecular mass (Mr) about 720000. Analysis on polyacrylamide gels containing sodium dodecyl sulphate reveals equal amounts of two different types of subunit, termed alpha (Mrabout 61000) and beta (Mrabout 60000); thus the oligomer has the composition a α6β6. The alpha and beta subunits have been separated; their amino-terminal sequences are different, and antibodies raised against one subunit do not cross-react with the other subunit. Antibodies raised against the binding protein do not cross-react with the Rubisco large subunit, but do cross-react with polypeptides ofMrabout 60000 in extracts of chloroplasts from wheat, barley and tobacco, and in extracts of leucoplasts from castor-bean endosperm. The binding protein is made as a higher-molecular-mass precursor whan leaf polysomes are translated in a wheatgerm extract containing chloramphenicol, but is not synthesized by isolated intact chloroplasts. Thus the binding protein subunits are synthesized by cytoplasmic ribosomes and hence are likely to be encoded by nuclear genes. EtiolatedPisumplants contain binding protein, but exposure to light does not cause the same dramatic increase in amount that is seen in the case of Rubisco. Treatment of stromal extracts with Mg-ATP in the range 0.1-5.0 mM causes dissociation of the binding protein oligomer into monomeric subunits; CTP, GTP, UTP, AMP and cyclic AMP do not have this effect. Mg2+is required for dissociation but can be replaced by Ca2+. Newly synthesized large subunits are released when the binding protein oligomer is dissociated, but re-attach when the dissociation is reversed by removal of ATP.

Purification and Properties of Ribulosebisphosphate Carboxylase Large Subunit Binding Protein

The large subunit binding protein, an abundant plastid protein implicated in the assembly of ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO), has been highly purified from leaves of Pisum sativum. The 720 kilodaltons purified binding protein is composed of two types of subunits of 60 and 61 kilodaltons. Highly specific polyclonal antibodies have been raised against the binding protein. The antibodies do not cross-react with the large subunit nor do anti-RubisCO antibodies cross-react with the binding protein. A higher molecular weight form of the binding protein is immunoprecipitated from products of P. sativum polysomes translated in a wheat-germ system, indicating that the binding protein is synthesized by cytoplasmic ribosomes. Immunoblotting reveals the presence of binding protein in extracts of tobacco, wheat and barley leaves and castor bean endosperm.The previously reported dissociation of the binding protein-large subunit complex upon addition of ATP in vitro has been confirmed and the fates of the dissociated subunits further investigated. The dissociated binding protein subunits are not phosphorylated or adenylated in vitro by added ATP.

ATP-released large subunits participate in the assembly of RuBP carboxylase.

Preincubation of 35S-methionine-labeled chloroplast extracts with ATP at 0 degree C potentiates the subsequent assembly of labeled large subunits into RuBPCase . This is correlated with the dissociation of newly synthesized large subunits from the 29S large subunit binding protein complex. These released large subunits then assemble into RuBPCase in a second, nucleotide-stimulated reaction. The data demonstrate that the 29S complex can play an active role in the assembly of RuBPCase .

The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro

Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.