Biomedical subjects

M Mann

Publications and source records attributed to M Mann.

At least 127 records · Page 7Linked to original sources

PubMed1998

Mtr10p functions as a nuclear import receptor for the mRNA-binding protein Npl3p.

MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85. Green fluorescent protein (GFP)-tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident. Purified Mtr10p forms a complex with Npl3p, an RNA-binding protein that shuttles in and out of the nucleus. In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)-containing protein is not. Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non-repetitive C-terminal tail. Mtr10p interacts in vitro with Gsp1p-GTP, but with low affinity. Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran-GTP plus RNA. This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran-GTP and newly synthesized mRNA.

Amino Acid Sequence↗

PubMed1998

Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins.

Entry into anaphase and exit from mitosis depend on a ubiquitin-protein ligase complex called the anaphase-promoting complex (APC) or cyclosome. At least 12 different subunits were detected in the purified particle from budding yeast, including the previously identified proteins Apc1p, Cdc16p, Cdc23p, Cdc26p, and Cdc27p. Five additional subunits purified in low nanogram amounts were identified by tandem mass spectrometric sequencing. Apc2p, Apc5p, and the RING-finger protein Apc11p are conserved from yeast to humans. Apc2p is similar to the cullin Cdc53p, which is a subunit of the ubiquitin-protein ligase complex SCFCdc4 required for the initiation of DNA replication.

Amino Acid Sequence↗

PubMed1998

The NOT proteins are part of the CCR4 transcriptional complex and affect gene expression both positively and negatively.

The CCR4 transcriptional regulatory complex consisting of CCR4, CAF1, DBF2 and other unidentified factors is one of several groups of proteins that affect gene expression. Using mass spectrometry, we have identified the 195, 185 and 116 kDa species which are part of the CCR4 complex. The 195 and 185 kDa proteins were found to be NOT1 and the 116 kDa species was identical to NOT3. NOT1, 2, 3 and 4 proteins are part of a regulatory complex that negatively affects transcription. All four NOT proteins were found to co-immunoprecipitate with CCR4 and CAF1, and NOT1 co-purified with CCR4 and CAF1 through three chromatographic steps in a complex estimated to be 1.2x10(6) Da in size. Mutations in the NOT genes affected many of the same genes and processes that are affected by defects in the CCR4 complex components, including reduction in ADH2 derepression, defective cell wall integrity and increased sensitivity to monoand divalent ions. Similarly, ccr4, caf1 and dbf2 alleles negatively regulated FUS1-lacZ expression, as do defects in the NOT genes. These results indicate that the NOT proteins are physically and functionally part of the CCR4 complex which forms a unique and novel complex that affects transcription both positively and negatively.

Alleles↗

PubMed1998

Nup116p and nup100p are interchangeable through a conserved motif which constitutes a docking site for the mRNA transport factor gle2p.

Nup116p and Nup100p are highly related yeast GLFG nucleoporins, but only Nup116p is stoichiometrically bound to Gle2p, a previously identified mRNA export factor. A short Gle2p-binding sequence within Nup116p (GLEBS; residues 110-166) is sufficient and necessary to anchor Gle2p at the nuclear pores, whereas the carboxy-terminal domain of Nup116p mediates its own nuclear pore complex (NPC) association. The GLEBS is evolutionarily conserved and found in rat/Xenopus Nup98 and an uncharacterized Caenorhabditis elegans ORF, but is absent from Nup100p. When the GLEBS is deleted from Nup116p, Gle2p dissociates from the nuclear envelope and clusters of herniated nuclear pores form. When the GLEBS is inserted into Nup100p, Nup100p-GLEBS complements both the thermosensitive and NPC-herniated phenotype of nup116- cells, and Gle2p is retargeted concomitantly to the NPCs. Thus, the in vivo function of Gle2p is strictly coupled to the short GLEBS within Nup116p which links this putative mRNA transport factor to the nuclear pores.

Amino Acid Sequence↗

PubMed1998

In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly.

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Rac-associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.

Actins↗

PubMed1998

Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification.

Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross-references demonstrated that there is no obvious pattern by which the mobility of a protein in one gel system can be used to predict its mobility in the other. Thus, as laboratories adopt the immobilized pH gradient-based 2-D gel systems, the only reliable means of translating the data gained with the carrier ampholyte-based gel system is to positively identify the proteins in both 2-D systems.

Ampholyte Mixtures↗

PubMed1998

Coexpression of G-CSF with an unglycosylated G-CSF receptor mutant results in secretion of a stable complex.

Previously, we have shown that the entire extracellular domain of the granulocyte-colony stimulating factor receptor (sG-CSFr) produced in Chinese hamster ovary (CHO) cells forms a stable complex with its ligand G-CSF, at a stoichiometry of 2:2. A truncated receptor molecule consisting of the cytokine receptor homology domain and N-terminus Ig-like domain (Ig CRH) behaves quite similarly. Both of these forms of the receptor are highly glycosylated. To address the importance of glycosylation toward receptor activity and stability, and possibly obtain nonglycosylated receptor for crystallization, mutations were made to replace four Asn residues which are N-glycosylated in the truncated receptor. Virtually no receptor was recovered from conditioned media of CHO cells transfected with this mutant construct, although a high-level of mRNA coding for receptor was detected; this mRNA was translated as determined by Western blots of cell lysates. These results indicate that the translated product is apparently not secreted from these cells. Cells transfected with mutant receptor cDNA were cotransfected with a cDNA construct expressing G-CSF in which the single O-glycosylation site was eliminated by mutation. Upon fermentation of the cotransfectants, we observed a large amount of receptor-ligand complex in the conditioned media. The purified unglycosylated complex appeared to be of the same binding stoichiometry and approximate binding affinity as that of complex formed by addition of purified ligand and unmutated receptor. These results show that while glycosylation of sG-CSFr is not necessary for ligand binding, it appears to be crucial in folding and export from the cell.

Animals↗

PubMed1998

Identifying proteins and post-translational modifications by mass spectrometry.

Major recent advances in hardware performance, sample-handling procedures and software algorithms now allow reliable and sensitive mass spectrometric identification of proteins. Mass spectrometry vastly outperforms traditional sequencing technologies and thereby greatly facilitates the elucidation of the functions of individual proteins as well as multiprotein complexes and larger protein assemblages.

Amino Acid Sequence↗

PubMed1998

Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure.

The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in preribosomal RNA (pre-rRNA) processing. The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demonstrated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine (psi) synthesis. To characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP complexes by anti-m3G-immunoaffinity and Mono-Q chromatography of Saccharomyces cerevisiae extracts. Sequence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are common to both the snR30 and snR42 complexes. Nhp2p is a highly basic protein that belongs to a family of putative RNA-binding proteins. Cbf5p has recently been demonstrated to be involved in ribosome biogenesis and also shows striking homology with known prokaryotic psi synthases. The presence of Cbf5p, a putative psi synthase in each H/ACA snoRNP suggests that this class of RNPs functions as individual modification enzymes. Immunoprecipitation studies using either anti-Cbf5p antibodies or a hemagglutinin-tagged Nhp2p demonstrated that both proteins are associated with all H/ACA-motif snoRNPs. In vivo depletion of Nhp2p results in a reduction in the steady-state levels of all H/ACA snoRNAs. Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a similar bipartite structure that we propose to be a major structural determining principle for all H/ACA snoRNPs.

Amino Acid Sequence↗

PubMed1998

Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex.

Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors--the spliceosome--is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.

Amino Acid Sequence↗

PubMed1998

A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins.

The U1 snRNP is essential for recognition of the pre-mRNA 5'-splice site and the subsequent assembly of the spliceosome. Yeast U1 snRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events. We have comprehensively analyzed the composition of yeast U1 snRNPs using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 snRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues. A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 snRNP, but its association is salt-sensitive. Therefore, we are unable to establish conclusively whether it binds specifically to the U1 snRNP. Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 snRNP has been demonstrated or proposed. We also show that the yeast SmB protein is a U1 snRNP component. Yeast U1 snRNP therefore contains 16 different proteins, including seven snRNP core proteins, three homologues of the metazoan U1 snRNP-specific proteins, and six yeast-specific U1 snRNP proteins. We have simultaneously continued the characterization of additional mutants isolated in a synthetic lethal (MUD) screen for genes that functionally cooperate with U1 snRNA. Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respectively. mud10 and mud15 affect the in vivo splicing efficiency of noncanonical introns. Moreover, mud10p strongly affects the in vitro formation of splicing complexes, and extracts from the mud15 strain contain a U1 snRNP that migrates aberrantly on native gels. Finally, we show that Nam8p/Mud15p contributes to the stability of U1 snRNP.

Amino Acid Sequence↗

PubMed1997

Actin: a target of lipopolysaccharide-induced phosphorylation in human monocytes.

We have previously reported that lipopolysaccharide (LPS) causes altered phosphate labelling of cytosolic proteins of 36 kDa and 38 kDa (p36/38) and that inhibition of phosphorylation is accompanied by a loss of cytokine production. Here we have purified the two phosphorylated proteins via two-dimensional polyacrylamide gel electrophoresis. P 36 was found to consist of two proteins p36a and p36b. The proteins were analysed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry and p36b was identified as gamma-actin, p36a as beta/gamma-actin. The ability of LPS to cause altered phosphate labelling of beta/gamma-actin suggests a participation of the microfilament network in LPS-induced monocyte activation.

Actins↗

PubMed1997

Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching.

Peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) followed by database searching with the set of measured peptide masses is now a powerful method for the identification of pure proteins. Protein mixtures--such as frequently occur due to comigration in polyacrylamide gel bands--have hitherto required protein sequencing. Here we demonstrate that such protein bands can also be analyzed by peptide mass mapping alone. Database searching with the complete list of peptide masses determined by delayed-extraction MALDI mass spectrometry with a mass error of less than 30 ppm retrieves the most prominent protein in a mixture. In a second step, the protein identity is further confirmed by matching as many of the measured peptide masses as possible to the retrieved amino acid sequence. Peptide masses remaining after this "second pass search" are searched again to identify the next component in the protein mixture. This iterative process is repeated until all major ion signals are accounted for. Protein mixtures consisting of two or more individual components in a single gel band can be analyzed, further increasing the general applicability of MALDI peptide mapping for protein identification.

Amino Acid Sequence↗

PubMed1997

The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'-->5' exoribonucleases.

We identified a complex in S. cerevisiae, the "exosome," consisting of the five essential proteins Rrp4p, Rrp41p, Rrp42p, Rrp43p, and Rrp44p (Dis3p). Remarkably, four of these proteins are homologous to characterized bacterial 3'-->5' exoribonucleases; Rrp44p is homologous to RNase II, while Rrp41p, Rrp42p, and Rrp43p are related to RNase PH. Recombinant Rrp4p, Rrp44p, and Rrp41p are 3'-->5' exoribonucleases in vitro that have distributive, processive, and phosphorolytic activities, respectively. All components of the exosome are required for 3' processing of the 5.8S rRNA. Human Rrp4p is found in a comparably sized complex, and expression of the hRRP4 gene in yeast complements the rrp4-1 mutation. We conclude that the exosome constitutes a highly conserved eukaryotic RNA processing complex.

Amino Acid Sequence↗

PubMed1997

Future horizons in cardiovascular molecular therapeutics.

In the past 2 decades, significant progress has been made in cardiovascular therapeutics. Effective drug therapies have been developed for hypertension, hypercholesterolemia, coronary artery disease, myocardial infarction, and congestive heart failure. Novel therapeutic strategies to treat cardiovascular disease consist of 3 major approaches: (1) changing the biology of vascular disease; (2) intervening in the ischemic event; or (3) modifying the post-ischemic course. The development of future therapies depends on continuing advancements in our understanding of the molecular mechanisms of vascular pathobiology. Novel therapies are aimed at the critical steps in vascular disease progression, which include reversing endothelial cell dysfunction, modulating thrombosis and inflammation, correcting dysregulated cell growth and apoptosis, modulating vascular phenotype, and modifying mechanicotransduction in vascular remodeling. Targeting these steps at the molecular level will stimulate the development of numerous therapeutic agents. Ongoing research will further define the role of the agents in the treatment of cardiovascular disease.

Animals↗

PubMed1997

IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

Cell Cycle Proteins↗

PubMed1997

Thrombomucin, a novel cell surface protein that defines thrombocytes and multipotent hematopoietic progenitors.

MEP21 is an avian antigen specifically expressed on the surface of Myb-Ets-transformed multipotent hematopoietic precursors (MEPs) and of normal thrombocytes. Using nanoelectrospray tandem mass spectrometry, we have sequenced and subsequently cloned the MEP21 cDNA and named the gene thrombomucin as it encodes a 571-amino acid protein with an extracellular domain typical of the mucin family of proteoglycans. Thrombomucin is distantly related to CD34, the best characterized and most used human hematopoietic stem cell marker. It is also highly homologous in its transmembrane/intracellular domain to podocalyxinlike protein-1, a rabbit cell surface glycoprotein of kidney podocytes. Single cell analysis of yolk sac cells from 3-d-old chick embryos revealed that thrombomucin is expressed on the surface of both lineage-restricted and multipotent progenitors. In the bone marrow, thrombomucin is also expressed on mono- and multipotent progenitors, showing an overlapping but distinct expression pattern from that of the receptor-type stem cell marker c-kit. These observations strengthen the notion that the Myb-Ets oncoprotein can induce the proliferation of thrombomucin-positive hematopoietic progenitors that have retained the capacity to differentiate along multiple lineages. They also suggest that thrombomucin and CD34 form a family of stem cell-specific proteins with possibly overlapping functions in early hematopoietic progenitors.

Animals↗

PubMed1997

A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function.

The small GTPase Rab5 plays an essential role in endocytic traffic. Rab GDP dissociation inhibitor delivers Rab5 to the membrane, where a nucleotide exchange activity allows recruitment of an effector protein, Rabaptin-5. Here we uncovered a novel 60 kDa Rab5-binding protein, Rabex-5. Rabex-5 forms a tight physical complex with Rabaptin-5, and this complex is essential for endocytic membrane fusion. Sequencing of mammalian Rabex-5 by nanoelectrospray mass spectrometry and cloning revealed striking homology to Vps9p, a yeast protein implicated in endocytic traffic. Rabex-5 displays GDP/GTP exchange activity on Rab5 upon delivery of the GTPase to the membrane. This demonstrates that a soluble exchange factor coupled to a Rab effector translocates from cytosol to the membrane, where the complex stabilizes the GTPase in the active state.

Animals↗