Formation Of Subunits Research Articles

The role of fcc tetrahedral subunits in the phase behavior of medium sized Lennard-Jones clusters

The free energy of a 600-atom Lennard-Jones cluster is calculated as a function of surface and bulk crystallinity in order to study the structural transformations that occur in the core of medium sized clusters. Within the order parameter range studied, we find the existence of two free energy minima at temperatures near freezing. One minimum, at low values of both bulk and surface order, belongs to the liquid phase. The second minimum exhibits a highly ordered core with a disordered surface and is related to structures containing a single fcc-tetrahedral subunit, with an edge length of seven atoms (l=7), located in the particle core. At lower temperatures, a third minimum appears at intermediate values of the bulk order parameter which is shown to be related to the formation of multiple l=6 tetrahedra in the core of the cluster. We also use molecular dynamics simulations to follow a series of nucleation events and find that the clusters freeze to structures containing l=5, 6, 7, and 8 sized tetrahedra as well as those containing no tetrahedral units. The structural correlations between bulk and surface order with the size of the tetrahedral units in the cluster core are examined. Finally, the relationships between the formation of fcc tetrahedral subunits in the core, the phase behavior of medium sized clusters and the nucleation of noncrystalline global structures such as icosahedra and decahedra are discussed.

Different cytoplasmic/nuclear distribution of S6 protein phosphorylated at S240/244 and S235/236

Higher eukaryotic ribosome biogenesis takes place in the nucleolus and requires the import of ribosomal proteins from the cytoplasm. The ribosomal protein S6 is essential for the formation of ribosome subunits, and in mice S6 heterozygosity triggers embryonal lethality. Downstream of the mTOR (mammalian target of rapamycin) and MAPK (mitogen-activated protein kinase) signalling pathways S6 protein is phosphorylated at clustered residues S235/236 and S240/244 upon numerous physiological and pathological stimuli. Here, we show that S240/244-phosphorylated S6 is predominantly nuclear but also detectable in the cytoplasm, whereas S235/236-phosphorylated S6 is almost exclusively localized to the nucleus of primary human cells and virtually undetectable in the cytoplasm. However, in transformed cells the latter can also be detected in the cytoplasm. Experiments with the mTOR inhibitor rapamycin revealed that neither blocking the phosphorylation of S6 at S235/236 and S240/244 nor arresting the cell cycle affects the cytoplasmic/nuclear localization of S6 protein. Our findings provide new insights into the regulation of S6 phosphorylation and S6 protein localization in mammalian cells.

Abstract 3159: RPS6 controls the translation of 5’ terminal oligopyrimidine tract-containing mRNAs through a physical interaction that regulates the loading of these messages on the polysome

Abstract The prevailing paradigm for mTOR signaling implicates RPS6 activation in translational control of mRNAs characterized by the presence of an oligopyrimidine tract (5’ terminal oligopyrimidine) within the 5’ untranslated region. These messages containing an oligopyrimidine tract are important for ribosomal biogenesis as they encode most of the translational apparatus. However, mechanistic details of how RPS6 is involved in the regulation of these oligopyrimidine tract-containing messages are still poorly understood. We demonstrated that through small inhibitory RNA (siRNA) knockdown of RPS6, 5’ TOP mRNA were selectively upregulated in heavy polysomal fractions. The ability of RPS6 to interact with endogenous mRNA containing 5’ TOP sequences was tested by immunoprecipitation (IP) analysis using lysates from Diffuse Large B-cell Lymphoma (DLBCL) cell lines (Farage and OCI-LY10) and a specific RPS6 antibody. The association of RPS6 with 5’ TOP mRNA was interrogated using RT-qPCR to examine mRNAs isolated from the IP material using primer sets specific for multiple 5’ TOP mRNAs. We found that 5’ TOP containing mRNA were highly enriched in IP material obtained with anti-RPS6 antibody compared with the background level of amplification seen in control immunoglobulin G (IgG) IP. We constructed a heterologous green fluorescent protein (GFP) mRNA containing the 5’ TOP sequence from the RPS6 gene in the 5’ UTR. Using mRNP-IP assays, RPS6 was found to interact with the heterologous construct containing the 5’ TOP sequence when compared to the normal GFP mRNA control. A number of previous publications indicated that aberrant control of protein translation contributes to lymphomagenesis. RPS6 has been shown to control the formation of nascent ribosomal subunits and to participate in ribosomal biogenesis. An overabundance of RPS6 has been postulated to lead to an increase in the total number of ribosomes and change the profile of translated mRNAs, including messages with a low affinity for the translation machinery i.e., oncogenic mRNAs. To investigate the possibility that RPS6 is overexpressed in lymphoma, we screened a panel of DLBCL samples for RPS6 protein levels. Utilizing immunohistochemistry, we observed that RPS6 protein levels were significantly increased in DLBCL compared to B cells found in the germinal center of matched normal reactive lymph nodes. Finally, using short hairpin RNA (shRNA) targeting RPS6 we demonstrated significantly increased apoptosis in DLBCL cell lines when compared to control shRNA. Our work sheds light on how RPS6 may regulate the translation of mRNA containing a 5’ TOP sequence through a direct protein-mRNA interaction and has potential therapeutic implications for targeting RPS6 in lymphoid malignancies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3159.

Reasons Why Aldehydes Do Not Generally Participate in Cucurbit[n]uril Forming Reactions

The reaction of substituted glycolurils or a glycoluril dimer with a dialdehyde (o-phthalaldehyde) delivers S-shaped dimers and an S-shaped tetramer selectively. A combination of X-ray crystallography, PM3 calculations, and product resubmission experiments establish that the S-shaped isomers are thermodynamically more stable than the C-shaped diastereomers which we attribute to the conformational preferences of the newly formed benzo bicyclo[3.3.2]decane ring system. The preferential formation of S-shaped subunits is one reason why o-phthalaldehyde and possibly other aldehydes do not usually participate in CB[n] forming reactions. We also present evidence that points toward an equilibrium between glycoluril monomer + phthalaldehyde and S-shaped dimer + water that responds to concentration over the 1-50 mM range. This result suggests a second reason, insufficient reactivity (e.g., low equilibrium constant), why o-phthalaldehyde and possibly other aldehydes do not participate in CB[n] forming reactions. Delineation of the reasons why some aldehydes fail in these reactions is the first step toward devising methods to overcome these limitations.

Solid-state transformation of hollow silica microspheres into hierarchical ZSM-5 having tunable mesopores

Mesoporous hierarchical ZSM-5 zeolites have been prepared via direct solid-state transformation of hollow silica gel microspheres without changing their spherical geometry. A two-stage hydrothermal method was used where tetrapropylammonium (TPA+) directed the formation of the MFI subunits in the first stage. In the second stage these subunits aggregated around the self-assembled cetyltrimethylammonium micelles and then crystallized by TPA+ templation. The mesoporous structure proved to be tunable in the pore size range of 3.2–4.8 nm by the addition of swelling agent 1,3,5-trimethylbenzene. Catalytic propene oligomerization showed less coke formed over mesoporous than microporous ZSM-5 catalysts.

The Ordered Transcription of RNA Domains Is Not Essential for Ribosome Biogenesis in Escherichia coli

Ribosome biogenesis is coupled to the transcription of ribosomal (r)RNAs, which enables an ordered and hierarchical assembly of the ribosome starting with the 5'-terminal domain. We constructed four circular permutants (CPs) of Escherichia coli rRNAs in which the original termini of 16S or 23S rRNAs were genetically connected, and new termini were created elsewhere within the same rRNAs (in helix 33 of 16S rRNA, or in helices 45, 63, and 78 of 23S rRNA). Unexpectedly, all CPs tested were able to rescue E. coli strain Delta7 prrn, which lacks all chromosomal rRNA operons. This result demonstrates that hierarchical assembly from the 5'-terminal domain of both 16S and 23S rRNAs is not essential for ribosomal formation in the cell. However, severe growth defects of all CPs were found in the absence of the DEAD box RNA helicase deaD, indicating that DeaD assists in the efficient assembly of each subunit in the cell.

Initiation of ribosome degradation during starvation in Escherichia coli

Ribosomes are known to be degraded under conditions of nutrient limitation. However, the mechanism by which a normally stable ribosome becomes a substrate for the degradation machinery has remained elusive. Here, we present in vitro and in vivo data demonstrating that free ribosome subunits are the actual targets of the degradative enzymes, whereas 70S particles are protected from such degradation. Conditions that increase the formation of subunits both in vitro and in vivo lead to enhanced degradation, while conditions favoring the presence of intact 70S ribosomes prevent or reduce breakdown. Thus, the simple formation of free 50S and 30S subunits is sufficient to serve as the initiation mechanism that allows endoribonuclease cleavage and subsequent ribosome breakdown.

Self‐Assembly of Fivefold‐Symmetric Molecules on a Threefold‐Symmetric Surface

Buckybowls: The adsorption of penta-tert-butylcorannulene, a molecule with fivefold symmetry, on Cu(111), a surface with threefold symmetry, is investigated by scanning tunneling microscopy complemented by structure calculations. The symmetry mismatch is resolved by the formation of threefold-symmetric subunits consisting of three molecules, which combine with single molecules to form a nearly perfect filling of the plane (see picture).

CK2β interacts with and regulates p21-activated kinases in Drosophila

The role of CK2β has been defined as the regulatory subunit of protein kinase CK2, which is a heterotetrameric complex composed of two CK2β and two catalytic active CK2α subunits. The identification of other serine/threonine kinases such as A-Raf, Chk1, and c-Mos that interact with and are regulated by CK2β has challenged this view and provided evidence for functions of CK2β outside the CK2 holoenzyme. In this report we describe the first interaction of Drosophila CK2β outside the CK2 holoenzyme with p21-activated kinase (PAK) proteins. This interaction is seen for distinct PAK and CK2β isoforms. In contrast to the CK2α–CK2β interaction, dimer formation of the CK2β subunits is not a prerequisite for binding of PAK proteins. Our results support the idea that CK2β can bind to PAK proteins in a CK2α independent manner and negatively regulates PAK kinase activity.

Assembly of the 30S Ribosomal Subunit.

Protein synthesis involves nearly a third of the total molecules in a typical bacterial cell. Within the cell, protein synthesis is performed by the ribosomes, and research over several decades has investigated ribosomal formation, structure, and function. This review provides an overview of the current understanding of the assembly of the Escherichia coli 30S ribosomal subunit. The E. coli 30S subunit contains one rRNA molecule (16S) and 21 ribosomal proteins (r-proteins; S1 to S21). The formation of functional subunits can occur as a self-assembly process in vitro; i.e., all the information required for the formation of active ribosomes resides in the primary sequences of the r-proteins and rRNAs. In vitro reconstitution of functional 30S subunits is carried out by using a mixture of TP30, individually purified natural or recombinant r-proteins, and natural 16S rRNA. Chemical probing and primer extension analysis have been used extensively to monitor changes in the reactivities of nucleotides in 16S rRNA during the in vitro reconstitution of 30S subunits. The potential roles for r-proteins in 30S subunit assembly were determined by omitting single proteins in reconstitution experiments. The RNPs resulting from single protein omissions were examined in terms of their composition and function to determine the roles of the absent proteins. Recent developments in understanding the structure of the 30S subunit have led to speculation about roles for some of the r-proteins in assembly. The crystal structures of the 30S subunit (1, 2) and the 70S ribosome (3) reveal details of the r-protein and rRNA interactions.

Effect of N-terminal Residues on the Structural Stability of Recombinant Horse L-chain Apoferritin in an Acidic Environment

The denaturation of recombinant horse L-chain apoferritin (rLF), which is composed of 24 L-chain subunits, in acidic solution was studied. Using two rLF mutants, lacking four (Fer4) or eight (Fer8) N-terminal amino acid residues, the effect of N-terminal residues on the protein's stability was investigated. Of the two mutants and wild-type rLF, the tertiary and secondary structures of Fer8 were found to be most sensitive to an acidic environment. The Fer8 protein dissociated easily into subunit dimers at or below pH 2.0. Comparing the crystal structures of the mutant proteins, deletion of the N-terminal residues was found to result in fewer inter- and intra-subunit hydrogen bonds. The loss of these bonds is assumed to be responsible for lower endurance against acidic denaturation in N-terminus-deleted mutants. These results indicated that the inter- and intra-subunit hydrogen bonds of N-terminal residues affect the denaturation, especially oligomer formation of apoferritin subunits and will be of use in designing ferritin-based nanodevices.

Chronic phencyclidine administration reduces the expression and editing of specific glutamate receptors in rat prefrontal cortex

Phencyclidine (PCP) induces a form of psychosis that mimics naturally occurring schizophrenia in the most relevant domains of the psychopathology. In this report, we investigated the effect of chronic treatment with PCP on expression and RNA editing of α-amino-propionic acid (AMPA) and kainate (KA) glutamate receptor (GluR), in the rat prefrontal cortex and the hippocampus. We found that chronic, but not acute, PCP treatment decreased GluRs expression in the rat prefrontal cortex but not in the hippocampus. In particular, the mRNA coding for GluR2 and GluR3 subunits were reduced by 50%, whereas those coding for KA GluR5 and GluR6 were decreased by 30%. In addition, we observed a decrease of the editing levels of the R/G site in the flop form of both GluR2 and GluR3 and a significant increase in the editing level of GluR6 Q/R site. The variation in the editing level of the R/G sites suggests that chronic PCP treatment induced the formation of glutamate receptor subunits with slower resensitization kinetics and, with respect to kainate receptors, an increase in the Q/R editing level might generate receptor channels with a lower permeability to cations. Combining all the data, it can be inferred that the PCP treatment induced a specific and site-selective reduction of glutamatergic neurotransmission in the prefrontal cortex but not in the hippocampus.

Effects of 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272) on Smooth Muscle Tone, Soluble Guanylyl Cyclase Activity, and NADPH Oxidase Activity/Expression in Corpus Cavernosum from Wild-Type, Neuronal, and Endothelial Nitric-Oxide Synthase Null Mice

We aimed to characterize the relaxation induced by the soluble guanylyl cyclase (sGC) stimulator 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and its pharmacological interactions with nitric oxide (NO) in the corpus cavernosum (CC) from wild-type (WT), endothelial nitric-oxide synthase (eNOS)(-/-), and neuronal (n)NOS(-/-) mice. The effect of BAY 41-2272 on superoxide formation and NADPH oxidase expression was also investigated. Tissues were mounted in myographs for isometric force recording. Enzyme immunoassay kits were used for cGMP determination. sGC activity was determined in the supernatant fractions of the cavernosal samples by the conversion of GTP to cGMP. Superoxide formation and expression of NADPH oxidase subunits were studied using the reduction of ferricytochrome c and Western blot analysis, respectively. BAY 41-2272 (0.01-10 microM) relaxed CC with pEC(50) values of 6.36 +/- 0.07 (WT), 6.27 +/- 0.06 (nNOS(-/-)), and 5.88 +/- 0.07 (eNOS(-/-)). The relaxations were accompanied by increases in cGMP levels. N(omega)-Nitro-L-arginine methyl ester inhibited BAY 41-2272-evoked responses in CC from WT and nNOS(-/-), but not eNOS(-/-).1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one reduced and sildenafil potentiated the relaxations induced by BAY 41-2272 in all groups. BAY 41-2272 enhanced NO (endogenous and exogenous)-induced relaxations in a concentration-dependent manner. Expression and activity of sGC was similar among the different groups. Superoxide formation was reduced by BAY 41-2272 (0.1-1 microM). The compound also inhibited p22(phox) and gp91(phox) expression induced by 9,11-dideoxy-11 alpha,9 alpha-epoxymethanoprostaglandin F(2 alpha (U46619). Our results demonstrated that sGC activation in the penis by BAY 41-2272 directly or via enhancement of NO effects may provide a novel treatment for erectile dysfunction, particularly in the event of an increased intrapenile oxidative stress.

Synthesis of ultrastable ordered mesoporous aluminosilicates molecular sieves with “hard template”

Ultrastable MSAMS-2 has been synthesized by the use of SBA-15 as “hard template” via twice re-crystallization both within the diluted solution of aluminosilicates sol–gel and glycerol. The ultrastable hydrothermal stability of MSAMS-2 might be ascribed to synergistic effect of the thicker wall, the higher condensation of silanol groups, the insertion of non-framework Al atoms and the formation of zeolites-like five-membered ring subunits within mesoporous framework.

Impaired ribosome biogenesis in Diamond-Blackfan anemia

AbstractThe gene encoding the ribosomal protein S19 (RPS19) is frequently mutated in Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. The consequence of these mutations on the onset of the disease remains obscure. Here, we show that RPS19 plays an essential role in biogenesis of the 40S small ribosomal subunit in human cells. Knockdown of RPS19 expression by siRNAs impairs 18S rRNA synthesis and formation of 40S subunits and induces apoptosis in HeLa cells. Pre-rRNA processing is altered, which leads to an arrest in the maturation of precursors to the 18S rRNA. Under these conditions, pre-40S particles are not exported to the cytoplasm and accumulate in the nucleoplasm of the cells in perinuclear dots. Consistently, we find that ribosome biogenesis and nucleolar organization is altered in skin fibroblasts from DBA patients bearing mutations in the RPS19 gene. In addition, maturation of the 18S rRNA is also perturbed in cells from a patient bearing no RPS19-related mutation. These results support the hypothesis that DBA is directly related to a defect in ribosome biogenesis and indicate that yet to be discovered DBA-related genes may be involved in the synthesis of the ribosomal subunits.

The insertion domain of the duck hepatitis B virus core protein plays a role in nucleocapsid assembly

Synthesis of hepadnaviral DNA is dependent upon both the viral DNA polymerase and the viral core protein, the subunit of the nucleocapsids in which viral DNA synthesis takes place. In a study of natural isolates of duck hepatitis B virus (DHBV), we cloned full-length viral genomes from a puna teal. One of the clones failed to direct viral DNA replication in transfected cells, apparently as a result of a 3 nt inframe deletion of histidine 107 in the core protein. Histidine 107 is located in the center of a predicted helical region of the “insertion domain”, a stretch of 45 amino acids which appears to be at the tip of a spike on the surface of the nucleocapsid. The mutation was introduced into a well-characterized strain of DHBV for further analysis. Core protein accumulated in cells transfected with the mutant DHBV but was partially degraded, suggesting that it was unstable. Assembled nucleocapsids were not detected by capsid gel electrophoresis. Interestingly, the mutant protein appeared to form chimeric nucleocapsids with wild-type core protein. The chimeric nucleocapsids supported viral DNA replication. These results suggest that the insertion domain of the spike may play a role either in assembly of stable nucleocapsids, possibly in formation of the dimer subunits, or in triggering nucleocapsid disintegration, required during initiation of new rounds of infection.

Controlled organization of ZnO building blocks into complex nanostructures

ZnO complex nanostructure with special mushroom-like morphology was prepared by hydrolysis of zinc acetate dehydrate (Zn(CH 3COO) 2⋅2H 2O) in water–methanol mixed solvent at 60 °C. The formation mechanism was studied using XRD investigation and FE-SEM observation, which showed that the mushroom-like particles were transformed from cauliflower-like layered basic zinc acetate (LBZA), Zn 5(OH) 8(CH 3COO) 2⋅2H 2O, and composed of ZnO subunits with average size less than 10 nm. The introduction of hexamethylenetetramine (HMTA, C 6H 12N 4) to the solution before deposition led to drastic changes in the morphologies of both aggregation particles and ZnO subunits. The novel ZnO microspheres, which were made of regular hexagonal plate-like ZnO with dimensional size 35 × 10 nm , were formed. These hexagonal plate-like ZnO subunits stacked very compactly and aligned regularly. Kinetic study of this unique complex nanostructure using TEM and FE-SEM observation showed the presence of HMTA played an important role on the formation of hexagonal ZnO subunits through different mechanisms related to the different parts of microspheres.

The Yeast Kinase Swe1 is Required for Proper Entry into Cell Cycle After Arrest Due to Ribosome Biogenesis and Protein Synthesis Defects

Sda1 is an essential protein required for cell cycle progression in Saccharomyces cerevisiae. Here, we show that the sda1-1 mutation causes a defect in the formation and nuclear export of 60S ribosomal subunits. Moreover, the sda1-1, but also other mutants defective in ribosome biogenesis (e.g., rix1-1 and tif6D), exhibit a G1 arrest, which could be the consequence of impaired ribosome biogenesis. Interestingly, additional deletion of the non-essential Swe1 kinase, the homolog of S. pombe Wee1, causes a pronounced delay in entering a new cell cycle in sda1-1, rix1-1 and tif6D cells, when shifted back from restrictive to permissive conditions. However, such a prolonged delay is independent of the Tyr19 phosphorylation in Cdc28. Moreover, the lack of Swe1 causes delay in budding and DNA replication in cells released from the G1 arrest due to the block of protein synthesis. Our data suggest that Swe1 is required for timely entry into cell cycle after a G1 arrest caused by impairment in pre-60S biogenesis and in protein synthesis. Therefore we propose that Swe1, which is required for coordination of cell growth and cell division in G2/M, also has a role in the beginning of the cell cycle.

Protein disulfide isomerase suppresses the transcriptional activity of NF-κB

We report here that the transcriptional activity of NF-κB is negatively regulated by protein disulfide isomerase (PDI). Over-expression of PDI in RAW 264.7 cells strongly suppressed the LPS-induced production of inflammatory cytokines as well as NF-κB-dependent luciferase activity. This negative regulation of NF-κB was reversed by bacitracin, a PDI inhibitor. Interestingly, NF-κB/DNA complex formation and phosphorylation of NF-κB subunits was intact in PDI-expressing cells following stimulation with LPS. In addition, PDI and another redox regulator, thioredoxin (TRX), had opposite effects on NF-κB-dependent gene expression: activation of the NF-κB pathway by TRX was suppressed by expression of PDI in a dose-dependent manner. Finally, PDI expression was induced by the anti-inflammatory cytokine IL-10, and IL-10-mediated inhibition of LPS-induced IL-6 expression was reduced by bacitracin. These findings clearly demonstrate that PDI is a negative regulator of NF-κB, and may act downstream of IL-10 in this signaling pathway.

Ribosomal proteins Rps0 and Rps21 of Saccharomyces cerevisiae have overlapping functions in the maturation of the 3' end of 18S rRNA.

The Rps0 proteins of Saccharomyces cerevisiae are components of the 40S ribosomal subunit required for maturation of the 3' end of 18S rRNA. Drosophila and human homologs of the Rps0 proteins physically interact with Rps21 proteins, and decreased expression of both proteins in Drosophila impairs control of cellular proliferation in hematopoietic organs during larval development. Here, we characterize the yeast RPS21A/B genes and show that strains where both genes are disrupted are not viable. Relative to the wild type, cells with disrupted RPS21A or RPS21B genes exhibit a reduction in growth rate, a decrease in free 40S subunits, an increase in the amount of free 60S subunits, and a decrease in polysome size. Ribosomal RNA processing studies reveal RPS21 and RPS0 mutants have virtually identical processing defects. The pattern of processing defects observed in RPS0 and RPS21 mutants is not a general characteristic of strains with suboptimal levels of small subunit ribosomal proteins, since disruption of the RPS18A or RPS18B genes results in related but distinct processing defects. Together, these data link the Rps0 and Rps21 proteins together functionally in promoting maturation of the 3' end of 18S rRNA and formation of active 40S ribosomal subunits.