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PubMed2004

Biological control through regulated transcriptional coactivators.

Gene activation in higher eukaryotes requires the concerted action of transcription factors and coactivator proteins. Coactivators exist in multiprotein complexes that dock on transcription factors and modify chromatin, allowing effective transcription to take place. While biological control focused at the level of the transcription factor is very common, it is now quite clear that a substantial component of gene control is directed at the expression of coactivators, involving pathways as diverse as B-cell development, smooth muscle differentiation, and hepatic gluconeogenesis. Quantitative control of coactivators allows the functional integration of multiple transcription factors and facilitates the formation of distinct biological programs. This coordination and acceleration of different steps in linked pathways has important kinetic considerations, enabling outputs of particular pathways to be increased far more than would otherwise be possible. These kinetic aspects suggest opportunities and concerns as coactivators become targets of therapeutic intervention.

Animals↗

PubMed2005

The proteasome: not just degrading anymore.

The proteasome is a large multiprotein complex that has a critical role in the degradation of ubiquitylated proteins. A fascinating paper in this issue of Cell (Lee et al., 2005) now reveals that the proteasome recruits the SAGA histone acetyltransferase complex to a target promoter during gene activation. This finding adds to the growing body of evidence indicating that the proteasome has nonproteolytic functions.

Gene Expression Regulation, Fungal↗

PubMed2006

Mind the GAP: Wnt steps onto the mTORC1 train.

The TSC1/2 tumor-suppressor complex controls protein synthesis through the regulation of mTOR. In this issue of Cell, Inoki et al. (2006) report that the kinases GSK3 and AMPK cooperate in the activation of TSC2 to inhibit mTOR activity. Surprisingly, the phosphorylation of TSC2 by GSK3 is markedly suppressed by Wnt signaling. This suggests that components of the mTOR pathway may be therapeutic targets for diseases linked to hyperactive Wnt signaling.

AMP-Activated Protein Kinases↗

PubMed2006

Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin.

The current view of canonical Wnt signalling is that following Wnt binding to its receptors (Frizzled-Lrp5/6), dishevelled (Dvl) becomes hyperphosphorylated, and the signal is transduced to the APC-GSK3beta-axin-beta-catenin multiprotein complex, which subsequently dissociates. As a result beta-catenin is not phosphorylated, escapes proteosomal degradation and activates its target genes after translocation to the nucleus. Here, we analyzed the importance of the Wnt-3a-induced phosphorylation and shift in electrophoretic migration of Dvl (PS-Dvl) for the activation of beta-catenin. Analysis of Wnt-3a time- and dose-responses in a dopaminergic cell line showed that beta-catenin is activated rapidly (within minutes) and at a low dose of Wnt-3a (1 ng/ml). Surprisingly, PS-Dvl appeared only after 30 min and at greater doses (> or =20 ng/ml) of Wnt-3a. Moreover, we found that a casein kinase 1 inhibitor (D4476) or siRNA for casein kinase 1 delta/epsilon (CK1delta/epsilon) blocked the Wnt-3a-induced PS-Dvl. Interestingly, CK1 inhibition or siRNA for CK1delta/epsilon did not ablate the activation of beta-catenin by Wnt-3a, indicating that there is a PS-Dvl-independent path to activate beta-catenin. The increase in beta-catenin activation by Wnt-3a (PS-Dvl-dependent or -independent) were blocked by Dickkopf1 (Dkk1), suggesting that the effect of Wnt-3a is in both cases mediated by Lrp5/6 receptors. Thus, our results show that Wnt-3a rapidly induce a partial activation of beta-catenin in the absence of PS-Dvl at low doses, while at high doses induce a full activation of beta-catenin in a PS-Dvl-dependent manner.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Beta strand peptidomimetics as potent PDZ domain ligands.

The search for general strategies for inhibiting protein-protein interactions has been stimulated by recognition of the key role they play in virtually every process of living systems. Multiprotein complex assembly and localization by PDZ domain-containing proteins exemplify processes critical to cell physiology and function that are mediated by beta strand association. Here we describe the development of substituted "@-tides," protease-resistant peptidomimetics incorporating conformationally restricted amino acid surrogates that reproduce the hydrogen-bonding pattern and side-chain functionality of a beta strand. The synthetic flexibility and generality of the substituted @-tide design was demonstrated by the synthesis of a panel of ligands for the alpha1-syntrophin PDZ domain. The rational design of a small molecule of unprecedented affinity for the PDZ domain suggests that these peptidomimetics may provide a general method for inhibiting protein-protein interactions involving extended peptide chains.

Biomimetics↗

PubMed2004

TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish.

TATA binding protein (TBP) is a key regulator of RNA polymerase transcription. It binds to core promoters, often in large multiprotein complexes, and nucleates RNA polymerase II (Pol II) transcription initiation. In addition to the previously described TBP-like factor present in metazoans (TLF/TRF2/TRP/TLP), we describe a third, vertebrate-specific member of the TBP protein family from zebrafish, called TBP2. Evolutionary conserved TBP2 homologs were also found in human, mouse, frog, and pufferfish. The N-terminal domains of TBP2s are divergent amongst themselves and different from those of TBPs; however, the core domain of TBP2s and TBPs are almost identical. TBP2 binds the TATA box, interacts with TFIIA and TFIIB (similarly to TBP), and can mediate Pol II transcription initiation. However, TBP2 shows contrasting expression patterns in the gonads and during embryonic development in comparison to TBP, suggesting differential function. Knockdown of zebrafish TBP2 results in specific reduction of the protein level, leading to a phenotype, which indicates the requirement of TBP2 for embryonic patterning. The presence of three different TBP family members in vertebrates suggests the existence of developmental stage- and tissue-specific preinitiation complexes with specific requirements for different TBP family members.

Animals↗

PubMed2006

Functional genomics identifies TOR-regulated genes that control growth and division.

BACKGROUND: The TOR (target of rapamycin) ser/thr protein kinase is the central component of a eukaryotic signaling pathway that regulates growth and is the direct target of the clinically useful drug rapamycin. Recent efforts have identified at least two multiprotein complexes that contain TOR, but little is known in higher eukaryotes about the genes downstream of TOR that control growth. RESULTS: By combining the use of a small molecule inhibitor (rapamycin), transcriptional profiling, and RNA interference in Drosophila tissue culture cells, we identified genes whose expression responds to Drosophila TOR (dTOR) inhibition and that regulate cell size. Several of the dTOR-regulated genes that function in cell size control have additional roles in cell division. Most of these genes are conserved in mammals and several are linked to human disease. This set of genes is highly enriched for regulators of ribosome biogenesis, which emphasizes the importance of TOR-dependent transcription in building the protein synthesis machinery in higher eukaryotes. In addition, we identify two dTOR-regulated genes, CG3071 and CG6677, whose human orthologs, SAW and ASH2L, are also under TOR-dependent transcriptional control and encode proteins with conserved functional roles in growth. CONCLUSIONS: We conclude that combining RNA interference with genomic analysis approaches, such as transcriptional profiling, is an effective way to identify genes functioning in a particular biological process. Moreover, this strategy, if applied in model systems with simpler genomes, can identify genes with conserved functions in mammals.

Animals↗

PubMed2006

mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that participates in at least two distinct multiprotein complexes, mTORC1 and mTORC2 . These complexes play important roles in the regulation of cell growth, proliferation, survival, and metabolism. mTORC2 is a hydrophobic motif kinase for the cell-survival protein Akt/PKB and, here, we identify mSin1 as a component of mTORC2 but not mTORC1. mSin1 is necessary for the assembly of mTORC2 and for its capacity to phosphorylate Akt/PKB. Alternative splicing generates at least five isoforms of the mSin1 protein , three of which assemble into mTORC2 to generate three distinct mTORC2s. Even though all mTORC2s can phosphorylate Akt/PKB in vitro, insulin regulates the activity of only two of them. Thus, we propose that cells contain several mTORC2 flavors that may phosphorylate Akt/PKB in response to different signals.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Polarized transport of Frizzled along the planar microtubule arrays in Drosophila wing epithelium.

Cells in a variety of developmental contexts sense extracellular cues that are given locally on their surfaces, and subsequently amplify the initial signal to achieve cell polarization. Drosophila wing cells acquire planar polarity along the proximal-distal (P-D) axis, in which the amplification of the presumptive cue involves assembly of a multiprotein complex that spans distal and proximal boundaries of adjacent cells. Here we pursue the mechanisms that place one of the components, Frizzled (Fz), at the distal side. Intracellular particles of GFP-tagged Fz moved preferentially toward distal boundaries before Fz::GFP and other components were tightly localized at the P/D cortex. Arrays of microtubules (MTs) were approximately oriented along the P-D axis and these MTs contributed to the formation of the cortical complex. Furthermore, there appeared to be a bias in the P-D MTs, with slightly more plus ends oriented distally. The hypothesis of polarized vesicular trafficking of Fz is discussed.

Adaptor Proteins, Signal Transducing↗

PubMed2006

mTORC2 Caught in a SINful Akt.

The target of rapamycin (TOR), a central controller of cell growth, is found in two distinct, highly conserved multiprotein complexes. Three recent papers in Cell (Jacinto et al., 2006), Developmental Cell (shiota et al., 2006; this issue), and Current Biology (Frias et al., 2006) shed light on mTOR complex 2 (mTORC2) composition and in vivo function. An important new finding is that mTORC2 determines Akt/PKB substrate specificity rather than absolute activity.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Identification of MMS19 domains with distinct functions in NER and transcription.

Nucleotide excision repair (NER) and RNA polymerase II (Pol II) transcription are essential cellular processes which are intimately intertwined. They share an indispensable multiprotein complex, TFIIH, and impairments in either process can impact the efficiency of the other. Like TFIIH, MMS19 is required for NER and Pol II transcription, but its precise role in each process is unknown. We showed previously that the human MMS19 gene originates multiple splice variants, some of which may encode distinct MMS19 protein isoforms. Here we characterize a novel MMS19 transcript and demonstrate for the first time that MMS19 splice variants are conserved across species and are functionally distinct. Expression of human MMS19 splice variants in mms19-deleted yeast cells produced unique patterns of thermosensitivity and ultraviolet radiation-sensitivity that point to three MMS19 structural domains with distinct in vivo functions. MMS19 polypeptides lacking domain A are able to fulfill the role of full-length MMS19 in NER but not in transcription. MMS19 polypeptides lacking part of domain B are efficient in transcription but not in NER. MMS19 polypeptides lacking domain C (HEAT repeats) are unable to fulfill either function. Our data suggest that the MMS19 HEAT repeat domain is essential for MMS19 function in NER and transcription, while domains A and B, within MMS19 N-terminus, modulate the balance between DNA repair and transcription. Our results highlight the functional significance of MMS19 transcripts and the possible contribution of MMS19 isoforms to regulate the switch between NER and transcription. Furthermore, our work associates for the first time specific protein domains with MMS19's role in NER and transcription.

Alternative Splicing↗

PubMed2006

Computer modelling in combination with in vitro studies reveals similar binding affinities of Drosophila Crumbs for the PDZ domains of Stardust and DmPar-6.

Formation of multiprotein complexes is a common theme to pattern a cell, thereby generating spatially and functionally distinct entities at specialised regions. Central components of these complexes are scaffold proteins, which contain several protein-protein interaction domains and provide a platform to recruit a variety of additional components. There is increasing evidence that protein complexes are dynamic structures and that their components can undergo various interactions depending on the cellular context. However, little is known so far about the factors regulating this behaviour. One evolutionarily conserved protein complex, which can be found both in Drosophila and mammalian epithelial cells, is composed of the transmembrane protein Crumbs/Crb3 and the scaffolding proteins Stardust/Pals1 and DPATJ/PATJ, respectively, and localises apically to the zonula adherens. Here we show by in vitro analysis that, similar as in vertebrates, the single PDZ domain of Drosophila DmPar-6 can bind to the four C-terminal amino acids (ERLI) of the transmembrane protein Crumbs. To further evaluate the binding capability of Crumbs to DmPar-6 and the MAGUK protein Stardust, analysis of the PDZ structural database and modelling of the interactions between the C-terminus of Crumbs and the PDZ domains of these two proteins were performed. The results suggest that both PDZ domains bind Crumbs with similar affinities. These data are supported by quantitative yeast two-hybrid interactions. In vivo analysis performed in cell cultures and in the Drosophila embryo show that the cytoplasmic domain of Crumbs can recruit DmPar-6 and DaPKC to the plasma membrane. The data presented here are discussed with respect to possible dynamic interactions between these proteins.

Animals↗

PubMed2005

Structure and functional role of supercomplexes of IsiA and Photosystem I in cyanobacterial photosynthesis.

Cyanobacteria express large quantities of the iron stress-inducible protein IsiA under iron deficiency. IsiA can assemble into numerous types of single or double rings surrounding Photosystem I. These supercomplexes are functional in light-harvesting, empty IsiA rings are effective energy dissipaters. Electron microscopy studies of these supercomplexes show that Photosystem I trimers bind 18 IsiA copies in a single ring, whereas monomers may bind up to 35 copies in two rings. Work on mutants indicates that the PsaF/J and PsaL subunits facilitate the formation of closed rings around Photosystem I monomers but are not obligatory components in the formation of Photosystem I-IsiA supercomplexes.

Bacterial Proteins↗

PubMed2005

Compartmentalisation of phosphodiesterases and protein kinase A: opposites attract.

Understanding the molecular organisation of intracellular signalling pathways is a topic of considerable research interest. Since many signalling enzymes are widely distributed and have several substrates, a critical component in signal transduction is the control of specificity. This is achieved, in part by the assembly of multiprotein complexes where clusters of signalling enzymes create focal points to disseminate the intracellular action of many hormones. This is particularly true for the cAMP dependent protein kinase (PKA) that is localised throughout the cell via its association with A-kinase anchoring proteins (AKAPs). Recent data suggest that some AKAPs also interact with phosphodiesterases (PDEs). Compartmentalisation of PDEs not only provides an elegant means to control PKA activation by monitoring the local cAMP flux, but also serves to concentrate and segregate the action of these important regulatory enzymes.

Animals↗

PubMed2005

Intimate relationship between the genes of two transcriptional coactivators, ADA2a and PIMT, of Drosophila.

PIMT, a transcriptional coactivator which interacts with and enhances nuclear receptor coactivator PRIP function, was identified recently in mammalian cells and suggested to function as a link between two major multiprotein complexes anchored by CBP/p300 and PBP. Here we describe that the gene of the Drosophila homologue of PIMT, designated as Dtl, is closely associated and has an overlapping promoter with a gene encoding another transcriptional coactivator, ADA2a, which in turn participates in GCN5 HAT-containing complexes. Ada2a also produces an RNA polII subunit, RPB4, via alternative splicing; consequently, an overlapping regulatory region serves for the production of three proteins, each involved in transcription. By studying expression of reporter gene fusions in tissue culture cells and transgenic animals we have demonstrated that the regulatory regions of Ada2a/Rpb4 and Dtl overlap and the Dtl promoter is partly within the Ada2a/Rpb4 coding region. The shared regulatory region contains a DRE element, binding site of DREF, the protein factor involved in the regulation of a number of genes which play a role in DNA replication and cell proliferation. Despite the perfectly symmetrical DRE, DREF seems to have a more decisive role in Ada2a/Rpb4 transcription than in the transcription of Dtl.

Acetyltransferases↗

PubMed2025

When glycobiology meets inflammasome activation: Insights and implications.

BACKGROUND: Glycobiology focuses mainly on the study of glycan structures and their biological functions. Glycans not only provide a basic energy supply through the tricarboxylic acid cycle and glycolysis but also serve as important immune regulators during pathogen invasion and homeostasis maintenance. Inflammasomes are critical multiprotein complexes of the immune system that detect both exogenous pathogenic threats and endogenous danger signals to mediate inflammatory responses. Glycobiology has revealed significant insights into the mechanisms of immune responses, particularly in the context of inflammasome activation. AIM OF REVIEW: This review summarizes the multifaceted relationships between glycobiology and inflammasome activation, highlighting how glycan structures, glycosylation patterns, and glycan-binding proteins influence inflammasome pathways. This review sheds light on novel targets for drug development aimed at modulating inflammatory pathways through the targeting of specific glycan structures. KEY SCIENTIFIC CONCEPTS OF REVIEW: Glycans directly or indirectly provide prime and activation signals for inflammasomes, glycosylation of inflammasome-related proteins by glycan structures modulates inflammasome activation and downstream inflammation, and the interaction between glycans and lectins also provides regulatory signals for inflammasome activation. This intersection of glycobiology and inflammasome activation presents a unique opportunity to elucidate the molecular mechanisms underlying inflammatory responses and their potential therapeutic implications.

Inflammasomes↗

PubMed2006

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment-protein complexes: peridinin-chlorophyll a protein (PCP) of Amphidinium carterae.

Peridinin-chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a "building block" approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving "optically dark" states of carotenoids in pigment-protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).

Amino Acids↗

PubMed2026

Epigenetic regulation of transgenes.

Gene therapy holds significant potential for treating genetic disorders, but the use of viral vectors is limited by factors such as immunogenicity, payload capacity, and high manufacturing costs. Nonviral gene delivery (NVGD) using plasmid DNA presents an attractive alternative; however, it typically provides a limited magnitude or duration of transgene expression. One potential reason for these shortcomings is the host cell's epigenetic regulation mechanisms, which can silence both viral and nonviral transgenes. Specifically, when foreign DNA enters the nucleus, it is detected by nuclear DNA sensors, such as IFI16, which initiate the assembly of a "restrictosome" or nuclear domain 10 (ND10) body. This multiprotein complex contains several components, such as PML, Speckled Proteins (e.g., SP100), DAXX, and ATRX that act as a scaffold for recruiting various epigenetic modifiers that subsequently deposit repressive histone modifications like H3K9me3 and H3K27me3 on the transgene chromatin. These marks induce DNA methylation and the subsequent condensation of plasmids or episomes into heterochromatin, which represses transgene expression. Alternatively, unmethylated CpG motifs in bacterial plasmid DNA can trigger innate immune responses in the cytosol, but this review will specifically focus on the detailed mechanisms of epigenetic regulation responsible for silencing plasmid DNA within the host cell nucleus. Addressing these nuclear defense mechanisms, potentially through strategies that manipulate DNA methylation or inhibit restrictosome activity, is crucial for advancing the development of safe, effective, and long-lasting plasmid viral and non-viral gene therapies.

Epigenesis, Genetic↗