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Biomedical subjects

M Mann

Publications and source records attributed to M Mann.

At least 55 records · Page 3Linked to original sources
PubMed2001

Analysis of proteins and proteomes by mass spectrometry.

A decade after the discovery of electrospray and matrix-assisted laser desorption ionization (MALDI), methods that finally allowed gentle ionization of large biomolecules, mass spectrometry has become a powerful tool in protein analysis and the key technology in the emerging field of proteomics. The success of mass spectrometry is driven both by innovative instrumentation designs, especially those operating on the time-of-flight or ion-trapping principles, and by large-scale biochemical strategies, which use mass spectrometry to detect the isolated proteins. Any human protein can now be identified directly from genome databases on the basis of minimal data derived by mass spectrometry. As has already happened in genomics, increased automation of sample handling, analysis, and the interpretation of results will generate an avalanche of qualitative and quantitative proteomic data. Protein-protein interactions can be analyzed directly by precipitation of a tagged bait followed by mass spectrometric identification of its binding partners. By these and similar strategies, entire protein complexes, signaling pathways, and whole organelles are being characterized. Posttranslational modifications remain difficult to analyze but are starting to yield to generic strategies.

Chromatography, Liquid↗
PubMed2001

Survey of pediatrician practices in retrieving statewide authorized newborn screening results.

OBJECTIVE: Mandated state newborn screening programs for the approximately 4 million infants born each year in the United States involves the following 5 components: 1) initial screening, 2) immediate follow-up testing of the screen-positive newborn, 3) diagnosis confirmation (true positive versus false positive), 4) immediate and long-term care, and 5) evaluation of all of the components of the system, including process and outcomes measures. Smooth functioning of this system requires pretest education of the parents as well as education and involvement of all health care providers who interact with the newborn screening system. Although extensive literature is available concerning public health aspects, technical standards/protocols, and discussion of the interfaces among the 5 components of the system, little information is available regarding physician awareness, involvement, and interactions with the system. The objective of this study was to determine, through a survey, primary care pediatricians' satisfaction with their state's newborn screening program. This was reflected in survey questions that asked how pediatricians were notified of the results of newborn screening tests that were performed on infants in their practice. METHODS: Two thousand questionnaires were sent to primary care pediatricians in all 50 states and the District of Columbia regarding their practices in retrieving statewide newborn screening results. Of the 2000 surveys, 574 (29%) responses from primary care pediatricians who care for at least 1 to 5 newborns each week form the basis of this report. Also reported are the commentaries of the physicians concerning their specific practices, overall assessment of the system, and ideas for improvement. RESULTS: Physicians reported their general satisfaction with the newborn screening system's ability to retrieve screen-positive infants for follow-up testing. However, communication and partnership with the primary care pediatrician regarding accessibility and timely retrieval of newborn screening test results was deemed less than optimal. Thirty-one percent of respondents indicated that notification for screen-positive test results was greater than 10 days, whereas 26% indicated that they do not receive the results of screen-negative tests and need to develop office procedures (contact birth hospital or state laboratory) to obtain results. Twenty-eight percent indicated that they do not actively seek results of newborn screening for their patients and presume that "no news is good news." Barriers to retrieving test results included that infants were born at hospitals where the physician does not have privileges, there were new transfers to the practice, infants were born in other states, personnel time was needed to track results, and there was a lack of a cohesive communication/reporting system that includes the primary care physician as an integral partner in the newborn screening communication process. Ninety-two percent of physicians would welcome an enhanced state system with direct communication to the primary care pediatrician as well as the birth hospital. CONCLUSION: Pediatricians recognize and endorse the benefits of newborn screening and believe that they play an important role in the efficient functioning of the system. An enhanced physician partnership with the newborn screening program will enable the timely follow-up of the screen-positive newborn for confirmatory testing. All test results need to be communicated to the pediatrician in a timely and efficient manner: 7 days for screen-positive results and 10 to 14 days for all results. Newborn screening test results of new patients who enter the practice should be available at the time of the first well-infant visit, ideally by 2 weeks of age. The majority of primary care pediatricians acknowledge the need to establish office protocols for the retrieval of newborn screening test results and would welcome an enhanced direct communication system with the state newborn screening program.

Attitude of Health Personnel↗
PubMed2000

Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways.

In an effort to clone novel tyrosine-phosphorylated substrates of the epidermal growth factor receptor, we have initiated an approach coupling affinity purification using anti-phosphotyrosine antibodies to mass spectrometry-based identification. Here, we report the identification of a signaling molecule containing a Src homology 3 domain as well as an immunoreceptor tyrosine-based activation motif (ITAM). This molecule is 55% identical to a previously isolated molecule designated signal transducing adaptor molecule (STAM) that was identified as an interleukin (IL)-2-induced phosphoprotein and is therefore designated STAM2. Tyrosine phosphorylation of STAM2 is induced by growth factors such as epidermal growth factor and platelet-derived growth factor as well as by cytokines like IL-3. Several of the deletion mutants tested except the one containing only the amino-terminal region underwent tyrosine phosphorylation upon growth factor stimulation, implying that STAM2 is phosphorylated on several tyrosine residues. STAM2 is downstream of the Jak family of kinases since coexpression of STAM2 with Jak1 or Jak2 but not an unrelated Tec family kinase, Etk, resulted in its tyrosine phosphorylation. In contrast to epidermal growth factor receptor-induced phosphorylation, this required the ITAM domain since mutants lacking this region did not undergo tyrosine phosphorylation. Finally, overexpression of wild type STAM2 led to an increase in IL-2-mediated induction of c-Myc promoter activation indicating that it potentiates cytokine receptor signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry.

Recently, we identified proteins that co-purify with the human spliceosome using mass spectrometry. One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5(+) gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The first catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The purified CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified. This work reports a first purification and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least five additional protein factors.

Adenosine Triphosphate↗
PubMed2000

Euplotes telomerase contains an La motif protein produced by apparent translational frameshifting.

Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in most eukaryotes. In the ciliate Euplotes aediculatus, the protein p43 biochemically co-purifies with active telomerase and appears to be stoichiometric with both the RNA and the catalytic protein subunit of this telomerase complex. Here we describe cloning of the gene for p43 and present evidence that it is an authentic component of the telomerase holoenzyme. Comparison of the nucleotide sequence of the cloned gene with peptide sequences of the protein suggests that production of full-length p43 relies on a programmed ribosomal frameshift, an extremely rare translational mechanism. Anti-p43 antibodies immunodeplete telomerase RNA and telomerase activity from E.aediculatus nuclear extracts, indicating that the vast majority of mature telomerase complexes in the cell are associated with p43. The sequence of p43 reveals similarity to the La autoantigen, an RNA-binding protein involved in maturation of RNA polymerase III transcripts, and recombinant p43 binds telomerase RNA in vitro. By analogy to other La proteins, p43 may function in chaperoning the assembly and/or facilitating nuclear retention of telomerase.

Amino Acid Motifs↗
PubMed2000

Tumor necrosis factor-induced microtubule stabilization mediated by hyperphosphorylated oncoprotein 18 promotes cell death.

Tumor necrosis factor (TNF)-induced cell death in the fibrosarcoma cell line L929 occurs independently of caspase activation and cytochrome c release. However, it is dependent on mitochondria and is characterized by increased production of reactive oxygen intermediates that are essential to the death process. To identify signaling molecules involved in this TNF-induced, reactive oxygen intermediate-dependent cell death pathway, we performed a comparative study by two-dimensional gel electrophoresis of phosphoproteins from a mitochondria-enriched fraction derived from TNF-treated and control cells. TNF induced rapid and persistent phosphorylation of the phosphorylation-responsive regulator of the microtubule (MT) dynamics, oncoprotein 18 (Op18). By using induced overexpression of wild type Op18 and phosphorylation site-deficient mutants S25A/S38A and S16A/S63A in L929 cells, we show that TNF-induced phosphorylation on each of the four Ser residues of Op18 promotes cell death and that Ser(16) and Ser(63) are the primary sites. This hyperphosphorylation of Op18 is known to completely turn off its MT-destabilizing activity. As a result, TNF treatment of L929 cells induced elongated and extremely tangled microtubules. These TNF-induced changes to the MT network were also observed in cells overexpressing wild type Op18 and, to a lesser extent, in cells overexpressing the S25A/S38A mutant. No changes in the MT network were observed upon TNF treatment of cells overexpressing the S16A/S63A mutant, and these cells were desensitized to TNF-induced cell death. These findings indicate that TNF-induced MT stabilization is mediated by hyperphosphorylation of Op18 and that this promotes cell death. The data suggest that Op18 and the MT network play a functional role in transduction of the cell death signal to the mitochondria.

Animals↗
PubMed2000

Functional genomics by mass spectrometry.

Systematic analysis of the function of genes can take place at the oligonucleotide or protein level. The latter has the advantage of being closest to function, since it is proteins that perform most of the reactions necessary for the cell. For most protein based ('proteomic') approaches to gene function, mass spectrometry is the method of choice. Mass spectrometry can now identify proteins with very high sensitivity and medium to high throughput. New instrumentation for the analysis of the proteome has been developed including a MALDI hybrid quadrupole time of flight instrument which combines advantages of the mass finger printing and peptide sequencing methods for protein identification. New approaches include the isotopic labeling of proteins to obtain accurate quantitative data by mass spectrometry, methods to analyze peptides derived from crude protein mixtures and approaches to analyze large numbers of intact proteins by mass spectrometry directly. Examples from this laboratory illustrate biological problem solving by modern mass spectrometric techniques. These include the analysis of the structure and function of the nucleolus and the analysis of signaling complexes.

Genome↗
PubMed2000

Nup116p associates with the Nup82p-Nsp1p-Nup159p nucleoporin complex.

Nup116p is a GLFG nucleoporin involved in RNA export processes. We show here that Nup116p physically interacts with the Nup82p-Nsp1p-Nup159p nuclear pore subcomplex, which plays a central role in nuclear mRNA export. For this association, a sequence within the C-terminal domain of Nup116p that includes the conserved nucleoporin RNA-binding motif was sufficient and necessary. Consistent with this biochemical interaction, protein A-Nup116p and the protein A-tagged Nup116p C-terminal domain, like the members of the Nup82p complex, localized to the cytoplasmic side of the nuclear pore complex, as revealed by immunogold labeling. Finally, synthetic lethal interactions were found between mutant alleles of NUP116 and all members of the Nup82p complex. Thus, Nup116p consists of three independent functional domains: 1) the C-terminal part interacts with the Nup82p complex; 2) the Gle2p-binding sequence interacts with Gle2p/Rae1p; and 3) the GLFG domain interacts with shuttling transport receptors such as karyopherin-beta family members.

Calcium-Binding Proteins↗
PubMed2000

The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex.

The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-protein ligase that targets cyclin B, securin and other destruction box containing proteins for proteolysis. Nine APC subunits have been identified in vertebrates and eleven in yeast, but for none of them it is known how they contribute to the catalysis of ubiquitination reactions. Here we report the mass spectrometric identification of CDC26 and of the RING-H2 finger protein APC11 in the human APC. We have expressed these proteins and several other APC subunits in Escherichia coli and have tested their activities in vitro. We find that APC11 alone is sufficient to allow the synthesis of multiubiquitin chains in the presence of E1 and UBC4. These multiubiquitin chains are partly unanchored and partly bound to APC11 itself. APC11 and UBC4 are also able to ubiquitinate securin and cyclin B, but these reactions show a decreased dependency on the destruction box. The integrity of the putative zinc binding RING-H2 finger is required for the ability of APC11 to support ubiquitination reactions. These results suggest that APC11 and UBC4 catalyze the formation of isopeptide bonds in APC-mediated ubiquitination reactions.

Amino Acid Sequence↗
PubMed2000

Use of mass spectrometry to study signaling pathways.

Activation of cells by extracellular stimuli, such as growth factors, initiates a cascade of events involving posttranslational modifications, including phosphorylation, formation of protein complexes, and induction or repression of gene expression. Traditionally, genetic methods or specific biochemical assays have been used to identify molecules involved in signaling pathways. Lately, mass spectrometry, combined with elegant biochemical approaches, has become a powerful tool for identifying proteins and posttranslational modifications. With this protocol, we hope to bridge the gap between the biochemical and molecular aspects of signal transduction pathways and the mass spectrometric tools and techniques that are available to study them. We provide methods for large-scale cell culture and immunoprecipitation of tyrosine-phosphorylated proteins, silver staining of gels, trypsin digests, and protein identification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and nanoelectrospray tandem mass spectrometry. We discuss the special requirements for the identification of phosphorylation sites in proteins by mass spectrometry. We describe enrichment of phosphopeptides from unseparated peptide mixtures by immobilized metal affinity column (IMAC) and the use of phosphatases to identify phosphorylated peptides. We also discuss specialized methods, such as precursor ion scanning in the negative mode and direct sequencing of phosphopeptides in the positive mode. Our goal is to provide detailed methods in use today for proteomic applications in general and for receptor-mediated signaling pathways in particular.

Animals↗
PubMed2000

A new variant of the gamma subunit of renal Na,K-ATPase. Identification by mass spectrometry, antibody binding, and expression in cultured cells.

The gamma subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney. On SDS-polyacryylamide gel electrophoresis, gamma runs as a doublet, but the origin and significance of the doublet is obscure. Mass spectrometry of the gamma chains of rat kidney Na, K-ATPase shows that gamma(a) (upper) has a mass of 7184.0 +/- 1 Da (carbamidomethyl cysteine), corresponding closely to that for the published sequence without the initiator methionine, while gamma(b) (lower) has a mass of 7337.9 +/- 1Da. Tryptic peptide mapping and sequencing by mass spectrometry reveals that the seven N-terminal residues of gamma(a), TELSANH, are replaced by Ac-MDRWYL in gamma(b), but otherwise the chains are identical. Antibodies raised against peptides TELSANHC and MDRWYLC recognize either gamma(a) or gamma(b) of the Na,K-ATPase, respectively. gamma(a) or gamma(b) cDNAs have been expressed in human embryonic kidney and HeLa cells. The major bands expressed correspond to gamma(a) or gamma(b) of renal Na, K-ATPase. Additional minor bands seen after transfection, namely gamma(a)' in human embryonic kidney and gamma(b)' in HeLa, are presumably cell-specific modifications. The present work clarifies earlier uncertainty regarding doublets seen in kidney and in transfected cells. In particular, the results show that renal Na, K-ATPase contains two variants of the gamma subunit with different sequences but otherwise are unmodified. We discuss the possible functional significance of the two variants.

Amino Acid Sequence↗
PubMed2000

Proteomics to study genes and genomes.

Proteomics, the large-scale analysis of proteins, will contribute greatly to our understanding of gene function in the post-genomic era. Proteomics can be divided into three main areas: (1) protein micro-characterization for large-scale identification of proteins and their post-translational modifications; (2) 'differential display' proteomics for comparison of protein levels with potential application in a wide range of diseases; and (3) studies of protein-protein interactions using techniques such as mass spectrometry or the yeast two-hybrid system. Because it is often difficult to predict the function of a protein based on homology to other proteins or even their three-dimensional structure, determination of components of a protein complex or of a cellular structure is central in functional analysis. This aspect of proteomic studies is perhaps the area of greatest promise. After the revolution in molecular biology exemplified by the ease of cloning by DNA methods, proteomics will add to our understanding of the biochemistry of proteins, processes and pathways for years to come.

Animals↗
PubMed2000

Identification of proteins in the postsynaptic density fraction by mass spectrometry.

Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. In this study, we have completed the identification of most major proteins in the PSD fraction with the use of an analytical method based on mass spectrometry coupled with searching of the protein sequence databases. At least one protein in each of 26 prominent protein bands from the PSD fraction has now been identified. We found 7 proteins not previously known to be constituents of the PSD fraction and 24 that had previously been associated with the PSD by other methods. The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mammalian homolog of the yeast septin protein cdc10, which is important for bud formation in yeast. Both myosin-Va and cdc10 are threefold to fivefold enriched in the PSD fraction over brain homogenates. Immunocytochemical localization of myosin-Va in cultured hippocampal neurons shows that it partially colocalizes with PSD-95 at synapses and is also diffusely localized in cell bodies, dendrites, and axons. Cdc10 has a punctate distribution in cell bodies and dendrites, with some of the puncta colocalizing with PSD-95. The results support a role for myosin-Va in transport of materials into spines and for septins in the formation or maintenance of spines.

Amino Acid Sequence↗
PubMed2000

The protein encoded by the proto-oncogene DEK changes the topology of chromatin and reduces the efficiency of DNA replication in a chromatin-specific manner.

The structure of chromatin regulates the genetic activity of the underlying DNA sequence. We report here that the protein encoded by the proto-oncogene DEK, which is involved in acute myelogenous leukemia, induces alterations of the superhelical density of DNA in chromatin. The change in topology is observed with chromatin but not with naked DNA and does not involve dissociation of core histones from chromatin. Moreover, these effects require histone H2A/H2B dimers in addition to histone H3/H4. We additionally tested whether the DEK protein affects DNA-utilizing processes and found that the DEK protein substantially reduces the replication efficiency of chromatin but not of naked DNA templates.

Blotting, Western↗
PubMed2000

Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation.

Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5' untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals.

Amino Acid Sequence↗