Biomedical subjects

Donald F Hunt

Publications and source records attributed to Donald F Hunt.

At least 73 records · Page 4Linked to original sources

PubMed2004

Identification of novel and widely expressed cancer/testis gene isoforms that elicit spontaneous cytotoxic T-lymphocyte reactivity to melanoma.

Multiple isoforms (TAG-1, TAG-2a, TAG-2b, and TAG-2c) of a novel cancer/testis antigen gene have been identified and are expressed in 84-88% of melanoma cell lines tested. The tumor antigen (TAG) genes are also expressed in K562, a myelogenous leukemia cell line, and they have homology to two chronic myelogenous leukemia-derived clones and a hepatocellular carcinoma clone in the human expressed sequence tags (EST) database, thus indicating that their expression is not restricted to melanomas. In contrast to the fact that many cancer/testis antigens are poorly immunogenic, the TAG-derived peptide, RLSNRLLLR, is recognized by HLA-A3-restricted, melanoma-specific CTLs that were obtained from a melanoma patient with spontaneous reactivity to the peptide. Unlike most cancer/testis antigen genes which are located on the X chromosome, the TAG genes are located on chromosome 5. The genes have the additional unusual features of being coded for in an open reading frame that is initiated by one of three nonstandard initiation codons, and the sequence coding the RLSNRLLLR peptide crosses an exon-exon boundary. The properties of the TAG antigens indicate that they are excellent vaccine candidates for the treatment of melanoma and perhaps other cancers.

Amino Acid Sequence↗

PubMed2004

The vertebrate Ndc80 complex contains Spc24 and Spc25 homologs, which are required to establish and maintain kinetochore-microtubule attachment.

How kinetochores bind to microtubules and move on the mitotic spindle remain unanswered questions. Multiple systems have implicated the Ndc80/Hec1 (Ndc80) kinetochore complex in kinetochore-microtubule interaction and spindle checkpoint activity. In budding yeast, Ndc80 copurifies with three additional interacting proteins: Nuf2, Spc24, and Spc25. Although functional vertebrate homologs of Ndc80 and Nuf2 exist, extensive sequence similarity searches have not uncovered homologs of Spc24 and Spc25. We have purified the xNdc80 complex to homogeneity from Xenopus egg extracts and identified two novel interacting proteins. Although the sequences have greatly diverged, we have concluded that these are the functional homologs of the yeast Spc24 and Spc25 proteins based on limited sequence similarity, common coiled-coil domains, kinetochore localization, similar phenotypes, and copurification with xNdc80 and xNuf2. Using both RNAi and antibody injection experiments, we have extended previous characterization of the complex and found that Spc24 and Spc25 are required not only to establish, but also to maintain kinetochore-microtubule attachments and metaphase alignment. In addition, we show that Spc24 and Spc25 are required for chromosomal movement to the spindle poles in anaphase.

Anaphase↗

PubMed2004

FANCG is phosphorylated at serines 383 and 387 during mitosis.

Fanconi anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and high incidence of leukemia and solid tumors. Eight genes have been cloned, with the accompanying protein products participating in at least two complexes, which appear to be functionally dependent upon one another. Previous studies have described chromatin localization of the FA core complex, except at mitosis, which is associated with phosphorylation of the FANCG protein (F. Qiao, A. Moss, and G. M. Kupfer, J. Biol. Chem. 276:23391-23396, 2001). The phosphorylation of FANCG at serine 7 by using mass spectrometry was previously mapped. The purpose of this study was to map the phosphorylation sites of FANCG at mitosis and to assess their functional importance. Reasoning that a potential kinase might be cdc2, which was previously reported to bind to FANCC, we showed that cdc2 chiefly phosphorylated a 14-kDa fragment of the C-terminal half of FANCG. Mass spectrometry analysis demonstrated that this fragment contains amino acids 374 to 504. Kinase motif analysis demonstrated that three amino acids in this fragment were leading candidates for phosphorylation. By using PCR-directed in vitro mutagenesis we mutated S383, S387, and T487 to alanine. Mutation of S383 and S387 abolished the phosphorylation of FANCG at mitosis. These results were confirmed by use of phosphospecific antibodies directed against phosphoserine 383 and phosphoserine 387. Furthermore, the ability to correct FA-G mutant cells of human or hamster (where S383 and S387 are conserved) origin was also impaired by these mutations, demonstrating the functional importance of these amino acids. S387A mutant abolished FANCG fusion protein phosphorylation by cdc2. The FA pathway, of which FANCG is a part, is highly regulated by a series of phosphorylation steps that are important to its overall function.

Animals↗

PubMed2003

MAPKAP kinase 2 phosphorylates tristetraprolin on in vivo sites including Ser178, a site required for 14-3-3 binding.

MAPKAP kinase 2 (MK2) is required for tumor necrosis factor synthesis. Tristetraprolin (TTP) binds to the 3'-untranslated region of tumor necrosis factor mRNA and regulates its fate. We identified in vitro and in vivo phosphorylation sites in TTP using nanoflow high pressure liquid chromatography microelectrospray ionization tandem mass spectrometry and novel methods for direct digestion of TTP bound to affinity matrices (GSH-beads or anti-Myc linked to magnetic beads). MK2Delta3B, activated in Escherichia coli by p38alpha, phosphorylates TTP in vitro at major sites Ser(52) and Ser(178) (>10-fold in abundance) as well as at several minor sites that were detected after enriching for phosphopeptides with immobilized metal affinity chromatography. MK2 phosphorylation of TTP creates a functional 14-3-3 binding site. In cells, TTP was phosphorylated at Ser(52), Ser(178), Thr(250), and Ser(316) and at SP sites in a cluster (Ser(80)/Ser(82)/Ser(85)). Anisomycin treatment of NIH 3T3 cells increased phosphorylation of Ser(52) and Ser(178). Overexpression of MK2 sufficed to increase phosphorylation of Ser(52) and Ser(178) but not Ser(80)/Ser(82)/Ser(85) or Thr(250). Thus, Ser(52) and Ser(178) are putative MK2 sites in vivo. Identified phosphosite(s) may be biologic switches controlling mRNA stability and translation.

14-3-3 Proteins↗

PubMed2003

Identification of a shared epitope recognized by melanoma-specific, HLA-A3-restricted cytotoxic T lymphocytes.

We previously established a melanoma-reactive cytotoxic T lymphocyte (CTL) line that recognizes multiple epitopes in the context of HLA-A3. To increase the number of peptides available for use in a vaccine for the treatment of melanoma, we identified one of these epitopes, SQNFPGSQK, through a combination of epitope reconstitution experiments and mass spectrometry. The SQNFPGSQK peptide was also found to be associated with HLA-A3 on an additional melanoma tumor line, thus indicating that the peptide is not unique to the melanoma tumor line from which it was isolated and thus, unlikely to arise through a mutational event. Although the protein origin of SQNFPGSQK has yet to be established, the shared nature of this epitope and the fact that it elicits a natural immune response indicates that it warrants further study to determine its usefulness as a vaccine component for the treatment of melanoma. The peptide may also be useful as a research tool for evaluating spontaneous anti-tumor immune responses in patients with melanoma.

Amino Acid Sequence↗

PubMed2003

Tapasin is a facilitator, not an editor, of class I MHC peptide binding.

Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin(+) and tapasin(-) cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin(-) cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.

Antigen Presentation↗

PubMed2003

Toward a protein profile of Escherichia coli: comparison to its transcription profile.

High-pressure liquid chromatography-tandem mass spectrometry was used to obtain a protein profile of Escherichia coli strain MG1655 grown in minimal medium with glycerol as the carbon source. By using cell lysate from only 3 x 108 cells, at least four different tryptic peptides were detected for each of 404 proteins in a short 4-h experiment. At least one peptide with a high reliability score was detected for 986 proteins. Because membrane proteins were underrepresented, a second experiment was performed with a preparation enriched in membranes. An additional 161 proteins were detected, of which from half to two-thirds were membrane proteins. Overall, 1,147 different E. coli proteins were identified, almost 4 times as many as had been identified previously by using other tools. The protein list was compared with the transcription profile obtained on Affymetrix GeneChips. Expression of 1,113 (97%) of the genes whose protein products were found was detected at the mRNA level. The arithmetic mean mRNA signal intensity for these genes was 3-fold higher than that for all 4,300 protein-coding genes of E. coli. Thus, GeneChip data confirmed the high reliability of the protein list, which contains about one-fourth of the proteins of E. coli. Detection of even those membrane proteins and proteins of undefined function that are encoded by the same operons (transcriptional units) encoding proteins on the list remained low.

Cell Membrane↗

PubMed2003

Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes.

Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic beta cells. CD8+ T cells are essential for this beta cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8+ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.

Animals↗

PubMed2003

Competition between two MHC binding registers in a single peptide processed from myelin basic protein influences tolerance and susceptibility to autoimmunity.

Experimental allergic encephalomyelitis (EAE) is an animal model for multiple sclerosis induced by stimulating myelin basic protein (MBP)-specific T cells. The MBP-specific repertoire in B10.PL mice is shaped by tolerance mechanisms that eliminate MBP121-150-specific T cells. In contrast, MBPAc1-11-specific T cells escape tolerance and constitute the encephalitogenic repertoire. To determine if this differential tolerance is caused by differences in the abundance of MBP epitopes generated by processing, MBP peptides were eluted from I-Au complexes and analyzed by mass spectrometry. Peptides were identified from both the NH2-terminal and MBP121-150 regions. Unexpectedly, MBPAc1-18 and Ac1-17, which contain the MBPAc1-11 epitope, were much more abundant than MBP121-150 peptides. The results demonstrate that competition between two I-Au binding registers, a low affinity register defined by MBPAc1-11 and a high affinity register defined by MBP5-16, prevents most of the NH2-terminal naturally processed peptides from binding in the MBPAc1-11 register. The small fraction of MBPAc1-18 bound in the MBPAc1-11 register is not sufficient to induce tolerance but provides a ligand for MBPAc1-11-specific T cells during disease. These results provide a basis for both the lack of tolerance to MBPAc1-11 and the ability of this epitope to become a target during autoimmunity.

Animals↗

PubMed2003

The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein.

Minor histocompatibility (H) antigens crucially affect the outcome of human leukocyte antigen (HLA)-identical allogeneic stem cell transplantation (SCT). To understand the basis of alloimmune responses against minor H antigens, identification of minor H peptides and their antigenicity-determining mechanisms is essential. Here we report the identification of HA-3 and its encoding gene. The HA-3 peptide, VTEPGTAQY (HA-3T), is encoded by the lymphoid blast crisis (Lbc) oncogene. We thus show for the first time that a leukemia-associated oncogene can give rise to immunogenic T-cell epitopes that may have participated in antihost and antileukemic alloimmune responses. Genotypic analysis of HA-3- individuals revealed the allelic counterpart VMEPGTAQY (HA-3M). Despite the lack of T-cell recognition of HA-3- cells, the Thr-->Met substitution had only a modest effect on peptide binding to HLA-A1 and a minimal impact on recognition by T cells when added exogenously to target cells. This substitution did not influence transporter associated with antigen processing (TAP) transport, but, in contrast to the HA-3T peptide, HA-3M is destroyed by proteasome-mediated digestion. Thus, the immunogenicity of minor H antigens can result from proteasome-mediated destruction of the negative allelic peptide.

A Kinase Anchor Proteins↗

PubMed2003

Weak agonist self-peptides promote selection and tuning of virus-specific T cells.

Recent progress has begun to define the interactions and signaling pathways that are triggered during positive selection. To identify and further examine self-peptides that can mediate positive selection, we searched a protein-database to find peptides that have minimal homology with the viral peptide (p33) that activates a defined P14 transgenic TCR. We identified four peptides that could bind the restriction element H-2D(b) and induce proliferation of P14 transgenic splenocytes at high concentration. Two of the four peptides (DBM and RPP) were able to positively select the virus-specific TCR in fetal thymic organ culture but were unable to induce clonal deletion. Reverse-phase HPLC and mass spectrometry demonstrated that these peptides were presented by H-2D(b) molecules on thymic epithelial cell lines. We also examined whether the selecting ligands altered T cell responsiveness in vitro. DBM-selected T cells lost their ability to respond to the positively selecting ligand DBM, whereas RPP-selected T cells only retained their ability to respond to high concentrations of RPP. These results demonstrate that self-peptides that mediate positive selection can differentially "tune" the activation threshold of T cells and alter the functional repertoire of T cells.

Amino Acid Sequence↗

PubMed2003

Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains.

The functional significance of mono-, di-, and trimethylation of lysine residues within histone proteins remains unclear. Antibodies developed to selectively recognize each of these methylated states at histone H3 lysine 9 (H3 Lys9) demonstrated that mono- and dimethylation localized specifically to silent domains within euchromatin. In contrast, trimethylated H3 Lys9 was enriched at pericentric heterochromatin. Enzymes known to methylate H3 Lys9 displayed remarkably different enzymatic properties in vivo. G9a was responsible for all detectable H3 Lys9 dimethylation and a significant amount of monomethylation within silent euchromatin. In contrast, Suv39h1 and Suv39h2 directed H3 Lys9 trimethylation specifically at pericentric heterochromatin. Thus, different methylated states of H3 Lys9 are directed by specific histone methyltransferases to "mark" distinct domains of silent chromatin.

Animals↗

PubMed2003

Bacterial biofilms of importance to medicine and bioterrorism: proteomic techniques to identify novel vaccine components and drug targets.

Biofilms are highly ordered microbial communities enmeshed in a carefully sculpted matrix designed for survival of organisms either in multi- or mono-genus/species in a specific microniche. In human disease, biofilm infections are some of the most recalcitrant to treat. Even with rigorous antibiotic regimens, some biofilms, such as those within the thick airway mucus of cystic fibrosis (CF) patients, persist throughout the course of the disease process. In this editorial, discussion will cover the utility of using advanced proteomic techniques to help identify potential weaknesses in the already impressive defensive armamentarium of biofilm bacteria. Two biofilm systems will be discussed herein, one of which is that of Pseudomonas aeruginosa biofilms within CF airway biofilms. The other is referred to as persistent 'bioterrorist agent biofilms' in which Francisella tularensis can grow on surfaces where environmental amoeba can phagocytose them, allowing for growth of F. tularensis within the amoebae.

Acanthamoeba↗

PubMed2002

Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation.

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.

Adenosine Triphosphatases↗

PubMed2002

Analysis of MHC class II antigen processing by quantitation of peptides that constitute nested sets.

Peptides associated with class II MHC molecules are of variable length because in contrast to peptides associated with class I MHC molecules, their amino and C termini are not constrained by the structure of the peptide interaction with the binding site. The proteolytic processing events that generate these peptides are still not well understood. To address this question, peptides extracted from HLA-DR*0401 were analyzed using two types of mass spectrometry instrumentation. This enabled identification of >700 candidate peptides in a single analysis and provided relative abundance information on 142 peptides contained in 11 nested sets of 3-36 members each. Peptides of 12 residues or less occurred only at low abundance, despite the fact that they were predicted to fully occupy the HLA-DR*0401 molecule in a single register. Conversely, the relative abundance of longer species suggested that proteolytic events occurring after MHC binding determine the final structure of most class II-associated peptides. Our data suggest that C-terminal residues of these peptides reflect the action of peptidases that cleave at preferred amino acids, while amino termini appear to be determined more by proximity to the class II MHC binding site. Thus, the analysis of abundance information for class II-associated peptides comprising nested sets has offered new insights into proteolytic processing of MHC class II-associated peptides.

Amino Acid Motifs↗

PubMed2002

A receptor for activated C kinase is part of messenger ribonucleoprotein complexes associated with polyA-mRNAs in neurons.

Long-lasting changes in synaptic functions after an appropriate stimulus require altered protein expression at the synapse. To restrict changes in protein composition to activated synapses, proteins may be synthesized locally as a result of transmitter receptor-triggered signaling pathways. Second messenger-controlled mechanisms that affect mRNA translation are essentially unknown. Here we report that a receptor for activated C kinase, RACK1, is a component of messenger ribonucleoprotein (mRNP) complexes. RACK1 is predominantly associated with polysome-bound, polyA-mRNAs that are being actively translated. We find it to be present in a complex with beta-tubulin and at least two mRNA-binding proteins, polyA-binding protein 1 and a 130 kDa polyA-mRNA binding protein (KIAA0217). Activation of PKCbeta2 in vitro by phosphatidylserine/diacylglycerol or in hippocampal slices by metabotropic glutamate receptor stimulation increased the amount of RACK1/PKCbeta2 associated with polysome-bound polyA-mRNAs. In vitro, PKCbeta2 can phosphorylate a subset of polyA-mRNA-associated proteins that are also phosphorylated under in vivo conditions. On the basis of these findings plus the somatodendritic localization of RACK1, we hypothesize that metabotropic glutamate receptor-triggered binding of activated PKCbeta2 to mRNP complexes bound to polyA-mRNAs is involved in activity-triggered control of protein synthesis.

Animals↗

PubMed2002

Gene silencing: trans-histone regulatory pathway in chromatin.

The fundamental unit of eukaryotic chromatin, the nucleosome, consists of genomic DNA wrapped around the conserved histone proteins H3, H2B, H2A and H4, all of which are variously modified at their amino- and carboxy-terminal tails to influence the dynamics of chromatin structure and function -- for example, conjugation of histone H2B with ubiquitin controls the outcome of methylation at a specific lysine residue (Lys 4) on histone H3, which regulates gene silencing in the yeast Saccharomyces cerevisiae. Here we show that ubiquitination of H2B is also necessary for the methylation of Lys 79 in H3, the only modification known to occur away from the histone tails, but that not all methylated lysines in H3 are regulated by this 'trans-histone' pathway because the methylation of Lys 36 in H3 is unaffected. Given that gene silencing is regulated by the methylation of Lys 4 and Lys 79 in histone H3, we suggest that H2B ubiquitination acts as a master switch that controls the site-selective histone methylation patterns responsible for this silencing.

Chromatin↗

PubMed2002

A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis.

Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago, its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested. Biochemical and genetic analyses suggest that U3 pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages. Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1 17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5' ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.

Cell Nucleolus↗