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PubMed1998

Participation of GRP94-related protein in secretion of pancreatic bile salt-dependent lipase and in its internalization by the intestinal epithelium.

In previous studies on the AR4-2J cell line, we have shown that secretion of bile salt-dependent lipase (BSDL) involves a multiprotein complex, including a protein of 94 kDa (p94) that is immunologically related to the chaperone Grp94, which seems to play essential roles in the folding process of BSDL. Combined biochemical and immunocytochemical investigations were carried out to study the secretion of BSDL by normal pancreatic cells and its transport to the small intestine where this enzyme is thought to exert its physiological function. Both BSDL and Grp94 antigenic sites were localized and found to be associated all along the pancreatic acinar cell secretory pathway. Grp94 and BSDL remain associated from leaving the pancreas until arriving at the intestinal lumen. In pancreatic juice, both proteins appear as a complex of high molecular mass (180 kDa) containing at least one each of p94 and BSDL molecules, interacting by hydrophobic forces. At the intestinal level, associated Grp94 and BSDL were detected on microvilli and in the endosomal compartment of enterocytes. The BSDL mRNA, however, was not expressed by the intestinal mucosa. The pancreatic Grp94-BSDL complex was internalized through the endosomal compartment of enterocytes. Finally, the two proteins dissociated in this compartment and BSDL, but not Grp94, was transferred to the basolateral membrane.

Animals↗

PubMed2002

Regulation of protrusive and contractile cell-matrix contacts.

The extracellular matrix is vital for tissue organisation in multicellular organisms. Cells attach to the extracellular matrix at discrete points on the cell surface, termed cell-matrix contacts. In general molecular terms, these contacts are assembled from large multiprotein complexes. However, many forms of matrix contacts can be distinguished by microscopy or by biochemical criteria, and these fulfil a diverse range of roles associated with cell adhesion, guidance, migration, matrix assembly, differentiation and survival. Two major functional categories are the protrusive and contractile matrix contacts. I describe contexts for the formation of protrusive or contractile contacts and discuss recent information on the molecular processes by which these contacts are specified, coordinated and regulated at a cellular level.

Animals↗

PubMed1995

Distribution of PCNA during postblastoderm cell division cycles in the Drosophila melanogaster embryo: effect of a string- mutation.

We used immunocytochemical methods and a specific antibody to identify proliferating cell nuclear antigen (PCNA) in Drosophila embryos during the postblastoderm cell division cycles. The strong nuclear staining observed during interphase disappeared at prophase, staining remained nil throughout the remaining mitotic (M) phases and reappeared in nuclei at the next interphase. As the embryos with the homozygous string- mutation sustained the strong staining signal in nuclei throughout embryonic development, disappearance of the staining signal probably depends on string function and is coupled with the onset of mitosis. When cells in embryos were arrested at M phase following treatment with TN-16 or Vinblastine, the staining signal with the anti-PCNA antibody was lost. However, Western blots showed that the level of PCNA protein in M phase-arrested embryos did not decrease. Therefore, the disappearance of staining signals is apparently due to reorganization of PCNA protein in the multiprotein complex in nuclei, rendering it inaccessible to the antibody, rather than to the degradation of PCNA protein. In contrast to findings in developing embryos, cultured Drosophila Kc cells stained strongly with the anti-PCNA antibody, during both interphase and the M phase. In genetic crossing experiments of transgenic flies carrying the lacZ gene under the control of the PCNA gene regulatory region (-607 to +137 with respect to the transcription initiation site) with string- mutant files, the PCNA gene promoter seems to function in a manner independent of cell cycle progression or of functions of the string gene.

Animals↗

PubMed2006

[Roles of nuclear receptors in the gene expression of drug-metabolizing enzymes under various physiological conditions].

The nuclear receptor constitutive androstane receptor (CAR), a key transcription factor for the expression of cytochrome P450 (CYP) 2B genes, resides in the cytoplasm under untreated conditions and translocates into the nucleus upon xenobiotic exposure. CAR forms a multiprotein complex including heat shock protein 90 in the cytoplasm as the glucocorticoid receptor, and it is likely that protein phosphatase 2A plays a critical role in the first step of CAR nuclear translocation. In addition to the xenobiotic induction of CYP2Bs, our recent studies have indicated that CAR is important for sex and strain differences and obesity/diabetes-associated changes in the expression of CYP2B genes. These results have raised the hypothesis that the expression of nuclear receptors varies depending on the physiologic condition, leading to the dysregulation of CYP expression. In obese mice fed a high-fat diet, however, hepatic CYP3A levels are drastically decreased without any significant changes in the expression of nuclear receptors including the pregnane X receptor and hepatocyte nuclear factor-4, which are known to be key transcription factors in the expression of CYP3A genes. These results indicate that it is important to investigate the mechanism of the transcriptional regulation of nuclear receptor genes as well as the activation of nuclear receptors to understand the CYP expression system fully.

Animals↗

PubMed2006

T. brucei RNA editing: action of the U-insertional TUTase within a U-deletion cycle.

Trypanosome RNA editing is massive post-transcriptional U-insertion and U-deletion, which generates mature mRNA coding regions through cycles of endonuclease, terminal U transferase (TUTase) or 3'-U-exo, and ligase action. Both types of editing are thought to be catalyzed by distinct sets of proteins of a multiprotein complex, and no enzymatic activity of wild-type editing complex had been shown to function in both forms of editing. By examining the individual steps of the U-deletion cycle using purified editing complex, traditional mitochondrial extract, and rapidly prepared cell lysate, we here demonstrate that TbMP57 TUTase of U-insertion can act efficiently within a U-deletion cycle. When physiological UTP levels are provided, it adds U's to the upstream cleavage fragment after U-deletional endonuclease and 3'-U-exo action, but before rejoining by the U-deletional ligase, generating partial U-deletion products. TUTase activity in U-deletion was not previously appreciated since its detection requires UTP, which is not normally added to in vitro U-deletion reactions. Fractionation and RNAi analyses show this U-addition in U-deletion requires TbMP57 TUTase be present and competent for U-insertion; such U-addition does not occur with another mitochondrial TUTase that is separate from the basic editing complex. Efficient TbMP57 action in both U-insertion and U-deletion suggests these two editing forms may be less separate than generally envisioned. Should such promiscuous TUTase action also occur in vivo, it could explain why editing utilizes substantially fewer U-deletional than U-insertional events and why partial editing appears preferential in U-deletion.

Animals↗

PubMed2006

KREPA4, an RNA binding protein essential for editosome integrity and survival of Trypanosoma brucei.

The 20S editosome, a multiprotein complex, catalyzes the editing of most mitochondrial mRNAs in trypanosomatids by uridylate insertion and deletion. RNAi mediated inactivation of expression of KREPA4 (previously TbMP24), a component of the 20S editosome, in procyclic form Trypanosoma brucei resulted in inhibition of cell growth, loss of RNA editing, and disappearance of 20S editosomes. Levels of MRP1 and REAP-1 proteins, which may have roles in editing but are not editosome components, were unaffected. Tagged KREPA4 protein is incorporated into 20S editosomes in vivo with no preference for either insertion or deletion subcomplexes. Consistent with its S1-like motif, recombinant KREPA4 protein binds synthetic gRNA with a preference for the 3' oligo (U) tail. These data suggest that KREPA4 is an RNA binding protein that may be specific for the gRNA Utail and also is important for 20S editosome stability.

Amino Acid Motifs↗

PubMed2006

Compositionally and functionally distinct editosomes in Trypanosoma brucei.

Uridylate insertion/deletion RNA editing in Trypanosoma brucei mitochondria is catalyzed by a multiprotein complex, the approximately 20S editosome. Editosomes purified via three related tagged RNase III proteins, KREN1 (KREPB1/TbMP90), KREPB2 (TbMP67), and KREN2 (KREPB3/TbMP61), had very similar but nonidentical protein compositions, and only the tagged member of these three RNase III proteins was identified in each respective complex. Three new editosome proteins were also identified in these complexes. Each tagged complex catalyzed both precleaved insertion and deletion editing in vitro. However, KREN1 complexes cleaved deletion but not insertion editing sites in vitro, and, conversely, KREN2 complexes cleaved insertion but not deletion editing sites. These specific nuclease activities were abolished by mutations in the putative RNase III catalytic domain of the respective proteins. Thus editosomes appear to be heterogeneous in composition with KREN1 complexes catalyzing cleavage of deletion sites and KREN2 complexes cleaving insertion sites while both can catalyze the U addition, U removal, and ligation steps of editing.

Amino Acid Sequence↗

PubMed2000

Structure and function of the HMGI(Y) family of architectural transcription factors.

The three known members of the HMGI(Y) family of high-mobility group (HMG) mammalian nonhistone nuclear proteins (HMG-I, HMG-Y, and HMGI-C) are thought to participate in numerous biological processes (transcription, replication, retroviral integration, genetic recombination, etc.) by virtue of their ability to recognize and alter the structure of both DNA and chromatin substrates. In vitro and in vivo the HMGI(Y) proteins preferentially bind to the narrow minor groove of stretches of AT-rich DNA by means of highly conserved peptide motifs called AT hooks. In vitro the HMGI(Y) proteins also have the ability to selectively bind to distorted DNA structures and to bend, unwind, and supercoil DNA substrates. Additionally, the HMGI(Y) proteins have the ability to interact with various protein transcription factors both in vitro and in vivo. Specific protein-DNA and protein-protein interactions permit the HMGI(Y) proteins to function as architectural transcription factors that regulate gene expression in vivo by controlling the formation of stereospecific multiprotein complexes on the AT-rich regions of certain gene promoters. Transcriptional overexpression of the HMGI(Y) genes is highly correlated with both cancerous transformation and increased metastatic potential of a number of different cancers, and chromosomal rearrangements involving AT-hook motifs have been associated with various types of benign human mesenchymal tumors. The levels of HMGI(Y) proteins in human cells have been proposed to be sensitive diagnostic indicators of both neoplastic transformation and metastatic progression. Drugs based on the AT-hook motif offer the potential for development of new tumor therapeutic reagents.

Amino Acid Sequence↗

PubMed2006

Structural organization of the 19S proteasome lid: insights from MS of intact complexes.

The 26S proteasome contains a 19S regulatory particle that selects and unfolds ubiquitinated substrates for degradation in the 20S catalytic particle. To date there are no high-resolution structures of the 19S assembly, nor of the lid or base subcomplexes that constitute the 19S. Mass spectra of the intact lid complex from Saccharomyces cerevisiae show that eight of the nine subunits are present stoichiometrically and that a stable tetrameric subcomplex forms in solution. Application of tandem mass spectrometry to the intact lid complex reveals the subunit architecture, while the coupling of a cross-linking approach identifies further interaction partners. Taking together our results with previous analyses we are able to construct a comprehensive interaction map. In summary, our findings allow us to identify a scaffold for the assembly of the particle and to propose a regulatory mechanism that prevents exposure of the active site until assembly is complete. More generally, the results highlight the potential of mass spectrometry to add crucial insight into the structural organization of an endogenous, wild-type complex.

COP9 Signalosome Complex↗

PubMed2004

Investigating the protein-protein interactions of the yeast Hsp90 chaperone system by two-hybrid analysis: potential uses and limitations of this approach.

The Hsp90 chaperone cycle involves sequential assembly of different Hsp90-containing multiprotein complexes, the accessory proteins ("cochaperones") that are associated with these complexes being exchanged as the cycle proceeds from its early to its late stages. To gain insight as to whether the 2-hybrid system could be used to probe the interactions of this Hsp90 system, yeast transformants were constructed that express the Gal4p deoxyribonucleic acid-binding domain (BD) fused to the 2 Hsp90 isoforms and the various Hsp90 system cochaperones of yeast. These "bait" fusions were then introduced by mating into other transformants expressing nearly all the 6000 proteins of yeast expressed as fusions to the Gal4p activation domain (AD). High throughput 2-hybrid screening revealed the ability of Hsp90 and Hsp90 system cochaperones to engage in stable interactions in vivo, both with each other and with the various other proteins of the yeast proteome. Consistent with the transience of most chaperone associations, interactions to Hsp90 itself were invariably weak and generally influenced by stress. Mutations within a Hsp90-BD bait fusion and an AD-Cdc37 "prey" fusion were used to provide in vivo confirmation of the in vitro data that shows that Cdc37p is interacting with the "relaxed" conformation of Hsp90 and also to provide indications that Cdc37p needs to be phosphorylated at its N-terminus for any appreciable interaction with Hsp90. A number of potentially novel cochaperone interactions were also identified, providing a framework for these to be analyzed further using other techniques.

Cell Cycle Proteins↗

PubMed2005

Reconstitution of endoplasmic reticulum-associated degradation using yeast membranes and cytosol.

The first compartment encountered by newly synthesized secreted proteins is the endoplasmic reticulum (ER). Before secreted proteins can traffic beyond the ER they must fold into their final conformations, and components of multiprotein complexes must assemble. Not surprisingly, then, the ER lumen houses a high concentration of molecular chaperones, factors that facilitate protein folding. However, if misfolded secreted proteins arise they may be selected and proteolyzed. This process, which removes potentially toxic proteins from the cell, has been termed ER-associated degradation (ERAD). Surprisingly, ERAD substrates are not degraded within the ER after being selected but are retrotranslocated to the cytoplasm and destroyed by the 26S proteasome. Thus, the ERAD pathway comprises substrate selection, substrate export from the ER, and substrate degradation, and each step in this pathway has been elucidated in part through an in vitro ERAD assay using reagents prepared from a model eukaryote, the yeast Saccharomyces cerevisiae. This chapter describes this in vitro ERAD assay in detail, and special considerations when performing the assay are noted.

Cell-Free System↗

PubMed2005

Heptaspanning membrane receptors and cytoskeletal/scaffolding proteins: focus on adenosine, dopamine, and metabotropic glutamate receptor function.

Most cellular functions are mediated by multiprotein complexes. In neurons, these complexes are directly involved in the proper neuronal transmission, which is responsible for phenomena like learning, memory, and development. In recent years studies based on two-hybrid screens and proteomic, biochemical, and cell biology approaches have shown that intracellular domains of G protein-coupled receptors (GPCRs) or heptaspanning membrane receptors (HSMRs) interact with intracellular proteins. These interactions are the basis of a protein network associated with these receptors, which includes scaffolding proteins containing one or several PDZ (postsynaptic-density-95/discs-large/zona occludens-1) domains, signaling proteins, and proteins of the cytoskeleton. The present article is focused on the emerging evidence for interactions of adenosine, dopamine, and metabotropic glutamate receptors, with scaffolding and cytoskeletal proteins that play a role in the targeting and anchoring of these receptors to the plasma membrane, thus contributing to neuronal development and plasticity. Finally, given the complexity of neurological disorders such as ischemic stroke, Alzheimer's disease, and epilepsy, exploitation of these HSMR-associated interactions might prove to be efficient in the treatment of such disorders.

Animals↗

PubMedDate not supplied

Mobilization of chromatin-bound Mcm proteins by micrococcal nuclease.

Mcm (minichromosome maintenance) proteins are important components of the eukaryotic replication initiation apparatus. We investigate the binding of human Mcm proteins to HeLa cell chromatin using micrococcal nuclease as a tool. In previous work we prepared chromatin under low ionic strength conditions. The use of a low salt buffer was necessary to prevent the dissociation of Mcm proteins. Here we use chromatin prepared at more physiological salt concentrations (100 mM NaCl) following the procedure of Fujita et al. (J. Biol. Chem. 272, 10928-10935; 1997) who had shown that ATP stabilizes the interaction of Mcm proteins with chromatin. We show here that micrococcal nuclease released Mcm proteins early during the digestion process suggesting that Mcm proteins reside on chromatin sites which are more open to nuclease attack than bulk chromatin. Released Mcm proteins sedimented through glycerol gradients as a multiprotein complex comprising several of the six known human Mcm proteins.

Adenosine Triphosphate↗

PubMed2005

The scaffold protein Homer1b/c links metabotropic glutamate receptor 5 to extracellular signal-regulated protein kinase cascades in neurons.

Group I metabotropic glutamate receptors (mGluRs) increase cellular levels of inositol-1,4,5-triphosphate (IP3) and thereby trigger intracellular Ca2+ release. Also, group I mGluRs are organized with members of Homer scaffold proteins into multiprotein complexes involved in postreceptor signaling. In this study, we investigated the relative importance of the IP3/Ca2+ signaling and novel Homer proteins in group I mGluR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in cultured rat striatal neurons. We found that selective activation of mGluR5, but not mGluR1, increased ERK1/2 phosphorylation. Whereas the IP3/Ca2+ cascade transmits a small portion of signals from mGluR5 to ERK1/2, the member of Homer family Homer1b/c forms a central signaling pathway linking mGluR5 to ERK1/2 in a Ca2+-independent manner. This was demonstrated by the findings that the mGluR5-mediated ERK1/2 phosphorylation was mostly reduced by a cell-permeable Tat-fusion peptide that selectively disrupted the interaction of mGluR5 with the Homer1b/c and by small interfering RNAs that selectively knocked down cellular levels of Homer1b/c proteins. Furthermore, ERK1/2, when only coactivated by both IP3/Ca2+- and Homer1b/c-dependent pathways, showed the ability to phosphorylate two transcription factors, Elk-1 and cAMP response element-binding protein, and thereby facilitated c-Fos expression. Together, we have identified two coordinated signaling pathways (a conventional IP3/Ca2+ vs a novel Homer pathway) that differentially mediate the mGluR5-ERK coupling in neurons. Both the Ca2+-dependent and -independent pathways are corequired to activate ERK1/2 to a level sufficient to achieve the mGluR5-dependent synapse-to-nucleus communication imperative for the transcriptional regulation.

Animals↗

PubMed2005

Drosophila target of rapamycin kinase functions as a multimer.

Target of rapamycin (TOR) is a conserved regulator of cell growth and metabolism that integrates energy, growth factor, and nutrient signals. The 280-kDa TOR protein functions as the catalytic component of two large multiprotein complexes and consists of an N-terminal HEAT-repeat domain and a C-terminal Ser/Thr kinase domain. Here we describe an allelic series of mutations in the Drosophila Tor gene and show that combinations of mutations in the HEAT and kinase domains of TOR display the rare genetic phenomenon of intragenic complementation, in which two or more defective proteins assemble to form a functional multimer. We present biochemical evidence that TOR self-associates in vivo and show that this multimerization is unaffected by positive or negative signals upstream of TOR. Consistent with multimerization of TOR, recessive mutations in the HEAT and kinase domains can dominantly interfere with wild-type TOR function in cells lacking TSC1 or TSC2. TOR multimerization thus partially accounts for the high apparent molecular weight of TOR complexes and offers novel therapeutic strategies for pathologies stemming from TOR hyperactivity.

Alleles↗

PubMed2004

Genetic defects in the oxidative phosphorylation (OXPHOS) system.

The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual genetic control. Due to this bigenomic control, the inheritance of OXPHOS system defects is either maternal, in the case of mitochondrial DNA mutations, autosomal or X-linked, in the case of nuclear gene defects. In this review, our current genetic understanding of OXPHOS system enzyme deficiencies will be summarized, and future directions that the field might take to unravel so-far genetically unresolved OXPHOS system enzyme deficiencies will be described, with special emphasis on complex I biogenesis.

DNA, Mitochondrial↗

PubMed2005

Mass spectrometry-based approaches to protein-ligand interactions.

One of the greatest current challenges in proteomics is to develop an understanding of cellular communication and regulation processes, most of which involve noncovalent interactions of proteins with various binding partners. Mass spectrometry plays an important role in all aspects of these research efforts. This article provides a survey of mass spectrometry-based approaches for exploring protein-ligand interactions. A wide array of techniques is available, and the choice of method depends on the specific problem at hand. For example, the high-throughput screening of compound libraries for binding to a specific receptor requires different approaches than structural studies on multiprotein complexes. This review is directed to readers wishing to obtain a concise yet comprehensive overview of existing experimental techniques. Specific emphasis is placed on emerging methods that have been developed within the last few years.

Animals↗

PubMed1997

Mammalian cell DNA replication.

The precise mechanisms involved in the regulation of the mammalian cell DNA-synthesizing machinery are poorly understood. In vitro DNA replication systems, in particular the employment of the simian virus 40 (SV40)-based cell-free DNA replication system, has identified several mammalian enzymes and proteins required for DNA synthesis. Although these proteins have been identified as playing a role in DNA replication, their functional organization allowing for the efficient replication of DNA has not been well defined. This review describes the proteins that have currently been defined as having a role in mammalian DNA replication and their proposed mechanisms of action. How these proteins may organize themselves to form multiprotein complexes, or larger DNA replication factories, allowing for efficient chromosomal DNA synthesis is discussed. In addition, the cell cycle regulation of mammalian DNA synthesis and the current status concerning mammalian DNA replication origins is described.

Animals↗