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PubMed2005

MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies.

Small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs) can silence target genes through several different effector mechanisms. Whereas siRNA-directed mRNA cleavage is increasingly understood, the mechanisms by which miRNAs repress protein synthesis are obscure. Recent studies have revealed the existence of specific cytoplasmic foci, referred to herein as processing bodies (P-bodies), which contain untranslated mRNAs and can serve as sites of mRNA degradation. Here we demonstrate that Argonaute proteins--the signature components of the RNA interference (RNAi) effector complex, RISC--localize to mammalian P-bodies. Moreover, reporter mRNAs that are targeted for translational repression by endogenous or exogenous miRNAs become concentrated in P-bodies in a miRNA-dependent manner. These results provide a link between miRNA function and mammalian P-bodies and suggest that translation repression by RISC delivers mRNAs to P-bodies, either as a cause or as a consequence of inhibiting protein synthesis.

3' Untranslated Regions↗
PubMed2002

Essential histidine and tryptophan residues in CcsA, a system II polytopic cytochrome c biogenesis protein.

Three distinct systems (I, II, and III) for catalysis of heme attachment to c-type apocytochromes are known. The CcsA and Ccs1 proteins are required in system II for the assembly of bacterial and plastid cytochromes c. A tryptophan-rich signature motif (WWD), also occurring in CcmC and CcmF found in system I, and three histidinyl residues, all strictly conserved in CcsA suggest a function in heme handling. Topological analysis of plastid CcsA in bacteria using the PhoA and LacZalpha reporters placed the WWD motif, the conserved residues His(212) and His(347) on the lumen side of the membrane, whereas His(309) was assigned a location on the stromal side. Functional analysis of CcsA through site-directed mutagenesis enabled the designation of the initiation codon of the ccsA gene and established the functional importance of the WWD signature motif and the absolute requirement of all three histidines for the assembly of plastid c-type cytochromes. In a ccsA mutant, a 200-kDa Ccs1-containing complex is absent from solubilized thylakoid membranes, suggesting that CcsA operates together with Ccs1. We propose a model where the WWD motif and histidine residues function in relaying heme from stroma to lumen and we postulate the existence of a cytochrome c assembly machinery containing CcsA, Ccs1 and additional components.

Amino Acid Motifs↗
PubMed2005

The signature amidase from Sulfolobus solfataricus belongs to the CX3C subgroup of enzymes cleaving both amides and nitriles. Ser195 and Cys145 are predicted to be the active site nucleophiles.

The signature amidase from the extremophile archeum Sulfolobus solfataricus is an enantioselective enzyme that cleaves S-amides. We report here that this enzyme also converts nitriles in the corresponding organic acid, similarly to the well characterized amidase from Rhodococcus rhodochrous J1. The archaeal and rhodococcal enzymes belong to the signature amidases and contain the typical serine-glycine rich motif. They work at different optimal temperature, share a high sequence similarity and both contain an additional CX3C motif. To explain their dual specificity, we built a 3D model of the structure of the S. solfataricus enzyme, which suggests that, in addition to the classical catalytic Ser-cisSer-Lys, a putative additional Cys-cisSer-Lys catalytic site, likely to be responsible for nitrile hydrolysis, is present in these proteins. The results of random and site-directed mutagenesis experiments, as well as inhibition studies support our hypothesis.

Amides↗
PubMed2002

Fine-tuning of the binding and dissociation of CO by the amino acids of the heme pocket of Coprinus cinereus peroxidase.

Resonance Raman and infrared spectra and the CO dissociation rates (k(off)) were measured in Coprinus cinereus peroxidase (CIP) and several mutants in the heme binding pocket. These mutants included the Asp245Asn, Arg51Leu, Arg51Gln, Arg51Asn, Arg51Lys, Phe54Trp, and Phe54Val mutants. Binding of CO to CIP produced different CO adducts at pH 6 and 10. At pH 6, the bound CO is H-bonded to the protonated distal His55 residue, whereas at alkaline pH, the vibrational signatures and the rate of CO dissociation indicate a distal side which is more open or flexible than in other plant peroxidases. The distal Arg51 residue is important in determining the rate of dissociation in the acid form, increasing by 8-17-fold in the Arg51 mutants compared to that for the wild-type protein. Replacement of the distal Phe with Trp created a new acid form characterized by vibrational frequencies and k(off) values very similar to those of cytochrome c peroxidase.

Amino Acid Substitution↗
PubMed2004

Function of the Trypanosome Argonaute 1 protein in RNA interference requires the N-terminal RGG domain and arginine 735 in the Piwi domain.

Argonaute proteins are central components of RNA interference (RNAi) and related phenomena in a wide variety of eukaryotes, including the early diverging protozoan Trypanosoma brucei. The single T. brucei Argonaute protein (TbAGO1) is in a complex with small interfering RNAs (siRNAs), and a fraction of this ribonucleoprotein particle is associated with polyribosomes. In this study, we generated a panel of insertion, deletion, and single point mutants of TbAGO1 and assayed them in vivo for their function in RNAi. In addition to the signature domains of Argonaute proteins, PAZ and Piwi, TbAGO1 has an N-terminal domain with a high abundance of RGG repeats. Deletion of the N-terminal domain blocked association of AGO1 with polyribosomes and severely affected mRNA cleavage. Nevertheless, the mutant protein was in a complex with siRNAs. In contrast, deletion of the Piwi domain led to a loss of siRNAs but did not abolish polyribosome association. Site-directed mutagenesis of conserved amino acids in the Piwi domain identified arginine 735 as essential for RNAi. Although the R735A mutant bound siRNAs and associated with polyribosomes, it displayed a severe defect in the cleavage of target mRNA.

Animals↗
PubMed2005

Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma.

Medulloblastoma is the most common malignant brain tumor in children. It is thought to result from the transformation of granule cell precursors (GCPs) in the developing cerebellum, but little is known about the early stages of the disease. Here, we identify a pre-neoplastic stage of medulloblastoma in patched heterozygous mice, a model of the human disease. We show that pre-neoplastic cells are present in the majority of patched mutants, although only 16% of these mice develop tumors. Pre-neoplastic cells, like tumor cells, exhibit activation of the Sonic hedgehog pathway and constitutive proliferation. Importantly, they also lack expression of the wild-type patched allele, suggesting that loss of patched is an early event in tumorigenesis. Although pre-neoplastic cells resemble GCPs and tumor cells in many respects, they have a distinct molecular signature. Genes that mark the pre-neoplastic stage include regulators of migration, apoptosis and differentiation, processes crucial for normal development but previously unrecognized for their role in medulloblastoma. The identification and molecular characterization of pre-neoplastic cells provides insight into the early steps in medulloblastoma formation, and may yield important markers for early detection and therapy of this disease.

Analysis of Variance↗
PubMed2006

A common haplotype of the glucokinase gene alters fasting glucose and birth weight: association in six studies and population-genetics analyses.

Fasting glucose is associated with future risk of type 2 diabetes and ischemic heart disease and is tightly regulated despite considerable variation in quantity, type, and timing of food intake. In pregnancy, maternal fasting glucose concentration is an important determinant of offspring birth weight. The key determinant of fasting glucose is the enzyme glucokinase (GCK). Rare mutations of GCK cause fasting hyperglycemia and alter birth weight. The extent to which common variation of GCK explains normal variation of fasting glucose and birth weight is not known. We aimed to comprehensively define the role of variation of GCK in determination of fasting glucose and birth weight, using a tagging SNP (tSNP) approach and studying 19,806 subjects from six population-based studies. Using 22 tSNPs, we showed that the variant rs1799884 is associated with fasting glucose at all ages in the normal population and exceeded genomewide levels of significance (P=10-9). rs3757840 was also highly significantly associated with fasting glucose (P=8x10-7), but haplotype analysis revealed that this is explained by linkage disequilibrium (r2=0.2) with rs1799884. A maternal A allele at rs1799884 was associated with a 32-g (95% confidence interval 11-53 g) increase in offspring birth weight (P=.002). Genetic variation influencing birth weight may have conferred a selective advantage in human populations. We performed extensive population-genetics analyses to look for evidence of recent positive natural selection on patterns of GCK variation. However, we found no strong signature of positive selection. In conclusion, a comprehensive analysis of common variation of the glucokinase gene shows that this is the first gene to be reproducibly associated with fasting glucose and fetal growth.

Adult↗
PubMed2004

Structure and mechanism of RNA ligase.

T4 RNA ligase 2 (Rnl2) exemplifies an RNA ligase family that includes the RNA editing ligases (RELs) of Trypanosoma and Leishmania. The Rnl2/REL enzymes are defined by essential signature residues and a unique C-terminal domain, which we show is essential for sealing of 3'-OH and 5'-PO4 RNA ends by Rnl2, but not for ligase adenylation or phosphodiester bond formation at a preadenylated AppRNA end. The N-terminal segment Rnl2(1-249) of the 334 aa Rnl2 protein comprises an autonomous adenylyltransferase/AppRNA ligase domain. We report the 1.9 A crystal structure of the ligase domain with AMP bound at the active site, which reveals a shared fold, catalytic mechanism, and evolutionary history for RNA ligases, DNA ligases, and mRNA capping enzymes.

Adenosine Monophosphate↗
PubMed2001

Carbon monoxide adducts of KatG and KatG(S315T) as probes of the heme site and isoniazid binding.

KatG, the catalase peroxidase from Mycobacterium tuberculosis, is important in the activation of the antitubercular drug, isoniazid. About 50% of isoniazid-resistant clinical isolates contain a mutation in KatG wherein the serine at position 315 is substituted with threonine, KatG(S315T). The heme pockets of KatG and KatG(S315T) and their interactions with isoniazid are probed using resonance Raman (rR) spectroscopy to characterize their ferrous CO complexes. Three vibrational modes, C-O and Fe-C stretching and Fe-CO bending, are assigned using 12CO and 13CO isotope shifts. Two conformers are observed for KatG-CO and KatG(S315T)-CO. Resonance Raman features assigned to form I are consistent with it having a neutral proximal histidine ligand and the Fe-C-O moiety hydrogen bonded to a distal residue. The nu(C-O) band for form I is sharp, consistent with a conformationally homogeneous Fe-CO unit. Form II also has a neutral proximal histidine ligand but is not hydrogen bonded. This appears to result in a conformationally disordered Fe-CO unit, as evidenced by a comparatively broad C-O stretching band. The 13CO-sensitive bands assigned to form II are predominant in the KatG(S315T)-CO rR spectrum. Isoniazid binding is apparent from the resonance Raman signatures of both WT KatG-CO and KatG(S315T)-CO. Moreover, isoniazid binding elicits an increase in the form I population of wild-type KatG-CO while having little, if any, effect on the already low population of form I of KatG(S315T)-CO. Since oxyKatG (compound III) also contains a low-spin diatomic ligand-heme adduct (heme-O2), it is reasonable to suggest that it too would exist as a mixture of conformers. Because the small form I population of KatG(S315T)-CO correlates with its inability to activate INH, we hypothesize that form I plays a role in INH activation.

Amino Acid Substitution↗
PubMed2000

Expression, purification, and characterization of HMWP2, a 229 kDa, six domain protein subunit of Yersiniabactin synthetase.

The six domain, 229 kDa HMWP2 subunit of the Yersinia pestis yersiniabactin (Ybt) synthetase has been expressed in soluble, full-length form in E. coli as a C-terminal His8 construct at low growth temperatures and with attenuated induction. All six domains of this nonribosomal peptide synthetase subunit, three phosphopantetheinylatable carrier protein domains (ArCP, PCP1, PCP2), one adenylation (A) domain, and two cyclization domains (Cy1, Cy2), have been assayed and are functional. Mutants that convert the phosphopantetheinylatable serine residue to alanine in each of the carrier protein domains accumulate acyl-S-enzyme intermediates upstream of the blocked apo carrier protein site. The ArCP mutant cannot be salicylated by the adenylation protein YbtE; the PCP1 mutant releases salicyl-cysteine from thiolysis of the Sal-S-ArCP intermediate; and the PCP2 mutant releases hydroxyphenyl-thiazolinyl-cysteine from the HPT-S-PCP1 acyl enzyme intermediate, all of which demonstrates processivity and directionality of chain growth. Restoration of the ArCP mutant's function was accomplished with the native ArCP fragment added in trans. The wild-type HMWP2 subunit accumulates hydroxyphenyl-4, 2-bithiazolinyl-S-enzyme at its most downstream PCP2 carrier site, presumably for transfer to the next subunit, HMWP1. The A domain was found to activate and transfer to PCP1 and PCP2 not only the natural L-Cys but also S-2-aminobutyrate, L-beta-chloroalanine, and L-Ser, enabling testing of the substrate specificity of the Cy domain. Probes of Cy domain function include mutagenesis of the Cy1 domain's conserved signature motif DX(4)DX(2)S to show that both D residues but not the S are crucial for both amide bond formation and heterocyclization. Also the Cy1 domain would accept an alternate upstream electrophilic donor substrate (2,3-dihydroxybenzoyl-S-ArCP) but would not process any of the three alternate downstream nucleophilic acceptors in place of Cys-S-PCP1, even for the amide bond-forming step in chain elongation.

Adenosine Triphosphate↗
PubMed2005

Impairment of the TFIIH-associated CDK-activating kinase selectively affects cell cycle-regulated gene expression in fission yeast.

The fission yeast Mcs6-Mcs2-Pmh1 complex, homologous to metazoan Cdk7-cyclin H-Mat1, has dual functions in cell division and transcription: as a partially redundant cyclin-dependent kinase (CDK)-activating kinase (CAK) that phosphorylates the major cell cycle CDK, Cdc2, on Thr-167; and as the RNA polymerase (Pol) II carboxyl-terminal domain (CTD) kinase associated with transcription factor (TF) IIH. We analyzed conditional mutants of mcs6 and pmh1, which activate Cdc2 normally but cannot complete cell division at restrictive temperature and arrest with decreased CTD phosphorylation. Transcriptional profiling by microarray hybridization revealed only modest effects on global gene expression: a one-third reduction in a severe mcs6 mutant after prolonged incubation at 36 degrees C. In contrast, a small subset of transcripts ( approximately 5%) decreased by more than twofold after Mcs6 complex function was compromised. The signature of repressed genes overlapped significantly with those of cell separation mutants sep10 and sep15. Sep10, a component of the Pol II Mediator complex, becomes essential in mcs6 or pmh1 mutant backgrounds. Moreover, transcripts dependent on the forkhead transcription factor Sep1, which are expressed coordinately during mitosis, were repressed in Mcs6 complex mutants, and Mcs6 also interacts genetically with Sep1. Thus, the Mcs6 complex, a direct activator of Cdc2, also influences the cell cycle transcriptional program, possibly through its TFIIH-associated kinase function.

Cell Cycle↗
PubMed1999

Guinea pig p53 mRNA: identification of new elements in coding and untranslated regions and their functional and evolutionary implications.

We report the sequence of the guinea pig p53 cDNA. The comparative analysis of the coding and noncoding regions of p53 cDNAs of all available complete vertebrate sequences has allowed us to single out new conserved signals possibly involved in p53 functional activity. We have focused our attention on the most variable region of the protein, the proline (P)-rich domain, suggested to play a fundamental role in antiproliferative pathways. In this domain we have identified the PXXXXP repeated motif and singled out a common consensus sequence that can be considered a signature for mammalian p53: PXXXXPX{0,4}PX{0,9}PA(T,P,I,)(S,P)WPL. We have demonstrated the significance of the PXXXXP motif in SH3-binding protein and suggested its structure to be a loop. Also, the 5' and 3' untranslated regions (UTRs) of the guinea pig were sequenced, and this study represents the first detailed structural analysis of the UTRs of the p53 mRNAs available in literature. The 5' UTR of guinea pig (233 nt) can be folded into a stable secondary structure resembling that predicted in mouse. The 3' UTR of guinea pig is 771 nt long and shows higher similarity with human than with rodent sequences, having a region of about 350 nt that is deleted in rat and mouse. In the 3' UTR we have identified the presence of a mammalian-wide interspersed repeat sequence and of a cytoplasmic polyadenylation element, which could be involved in translational activation by promoting polyadenylation of mRNA, providing information about a possible mechanism of regulation of p53 expression mediated by the 3' UTR of the mRNA. The observations presented here could open new avenues to targeted mutations and experimental approaches useful in investigating new regulation mechanisms of p53 translation, activity, and stability.

3' Untranslated Regions↗
PubMed2025

A small cationic probe for accurate, punctate discovery of RNA tertiary structure.

RNA molecules fold into intricate three-dimensional tertiary structures that are central to their biological functions. Yet reliably discovering new motifs that form true tertiary interactions remains a major challenge. Here we show that RNA tertiary folding occasionally generates electronegative motifs that react selectively with the small, positively-charged probe trimethyloxonium (TMO). Sites with enhanced reactivity to TMO, compared with the neutral reagent dimethyl sulfate (DMS), are indicative of tertiary structure and define T-sites. These positions share a structural signature in which a reactive nucleobase is adjacent to non-bridging phosphate oxygens, creating localized regions of negative charge. T-sites consistently map to the cores of higher-order structural interactions and functional centers across diverse RNAs, including distinct states in conformational ensembles. In the 10,723-nt dengue virus genome, three strong T-sites were detected, each within a complex structure required for viral replication. Cation-based covalent chemistry enables high-confidence discovery and analysis of functional RNA tertiary motifs across long and complex RNAs, opening new opportunities for transcriptome-wide structural analysis.

RNA electrostatics↗
PubMed1997

Lipoprotein from the osmoregulated ABC transport system OpuA of Bacillus subtilis: purification of the glycine betaine binding protein and characterization of a functional lipidless mutant.

The OpuA transport system of Bacillus subtilis functions as a high-affinity uptake system for the osmoprotectant glycine betaine. It is a member of the ABC transporter superfamily and consists of an ATPase (OpuAA), an integral membrane protein (OpuAB), and a hydrophilic polypeptide (OpuAC) that shows the signature sequence of lipoproteins (B. Kempf and E. Bremer, J. Biol. Chem. 270:16701-16713, 1995). The OpuAC protein might thus serve as an extracellular substrate binding protein anchored in the cytoplasmic membrane via a lipid modification at an amino-terminal cysteine residue. A malE-opuAC hybrid gene was constructed and used to purify a lipidless OpuAC protein. The purified protein bound radiolabeled glycine betaine avidly and exhibited a KD of 6 microM for this ligand, demonstrating that OpuAC indeed functions as the substrate binding protein for the B. subtilis OpuA system. We have selectively expressed the opuAC gene under T7 phi10 control in Escherichia coli and have demonstrated through its metabolic labeling with [3H]palmitic acid that OpuAC is a lipoprotein. A mutant expressing an OpuAC protein in which the amino-terminal cysteine residue was changed to an alanine (OpuAC-3) was constructed by oligonucleotide site-directed mutagenesis. The OpuAC-3 protein was not acylated by [3H]palmitic acid, and part of it was secreted into the periplasmic space of E. coli, where it could be released from the cells by cold osmotic shock. The opuAC-3 mutation was recombined into an otherwise wild-type opuA operon in the chromosome of B. subtilis. Unexpectedly, this mutant OpuAC system still functioned efficiently for glycine betaine acquisition in vivo under high-osmolarity growth conditions. In addition, the mutant OpuA transporter exhibited kinetic parameters similar to that of the wild-type system. Our data suggest that the lipidless OpuAC-3 protein is held in the cytoplasmic membrane of B. subtilis via its uncleaved hydrophobic signal peptide.

ATP-Binding Cassette Transporters↗
PubMed2001

Characterization and manipulation of the acyl chain selectivity of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme that catabolizes several neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. FAAH belongs to a large group of hydrolytic enzymes termed the amidase signature (AS) family that is defined by a conserved, linear AS sequence of approximately 130 amino acids. Members of the AS family display strikingly different substrate selectivities, yet the primary structural regions responsible for defining substrate recognition in these enzymes remain unknown. In this study, a series of unbranched p-nitroanilide (pNA) substrates ranging from 6 to 20 carbons in length was used to probe the acyl chain binding specificity of FAAH, revealing that this enzyme exhibits a strong preference for acyl chains 9 carbons in length or longer. A fluorophosphonate inhibitor of FAAH containing a photoactivatable benzophenone group was synthesized and used to locate a region of the enzyme implicated in substrate binding. Protease digestion and mass spectrometry analysis of FAAH-inhibitor conjugates identified the major site of cross-linking as residues 487-493. Site-directed mutagenesis revealed that a single residue in this region, I491, strongly influenced substrate specificity of FAAH. For example, an I491A mutant displayed a greatly reduced binding affinity for medium-chain pNA substrates (7-12 carbons) but maintained nearly wild-type binding and catalytic constants for longer chain substrates (14-20 carbons). Mutation of I491 to aromatic or more polar residues generated enzymes with relative hydrolytic efficiencies for medium- versus long-chain pNAs that varied up to 90-fold. Collectively, these studies indicate that I491 participates in hydrophobic binding interactions with medium-chain FAAH substrates. Additionally, the significant changes in substrate selectivity achieved by single amino acid changes suggest that FAAH possesses a rather malleable substrate binding domain and may serve, along with other AS enzymes, as a template for the engineering of amidases with novel and/or tailored specificities.

Alanine↗
PubMed2003

Chromatin as a tool for the study of genome function in cancer.

The transition to malignancy requires an extensive reconfiguration of the genome's expression program that does not result entirely from actual changes in primary DNA sequence-i.e., mutation. Epigenetic-meta-DNA-gene expression states result from an assembly over a given locus of a poorly understood nucleoprotein entity that includes histones and other architectural components of chromatin, nonhistone DNA-bound regulators, and additional chromatin-bound polypeptides. This structure is rapidly reestablished in the wake of the DNA replication fork, thus ensuring its persistence in rapidly proliferating cells and thereby yielding an exceptionally stable mode of gene expression. Chromatin is the perfect vehicle for enabling such genome control. During S phase both covalently modified histones and histone-associated regulatory proteins distribute to the newly synthesized daughter chromatids in a form of "molecular dowry" inherited from the G(1) state of the genome, and impose a specific mode of function on the underlying DNA. An extensively studied example of chromatin-based epigenetic inheritance connects DNA methylation to the targeting of chromatin remodeling and modification. In a broad sense, however, genome reprogramming in cancer is associated with the remodeling of a multitude of regulatory DNA stretches-e.g., promoters, enhancers, locus control regions (LCRs), insulators, etc.-into a specific chromatin architecture. This architectural entity provides a general molecular signature of the cancer epigenome that complements and significantly expands its DNA methylation-based component.

Chromatin↗
PubMed2006

Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling.

Individuals with Down syndrome (DS) are predisposed to develop acute megakaryoblastic leukemia (AMKL), characterized by expression of truncated GATA1 transcription factor protein (GATA1s) due to somatic mutation. The treatment outcome for DS-AMKL is more favorable than for AMKL in non-DS patients. To gain insight into gene expression differences in AMKL, we compared 24 DS and 39 non-DS AMKL samples. We found that non-DS-AMKL samples cluster in two groups, characterized by differences in expression of HOX/TALE family members. Both of these groups are distinct from DS-AMKL, independent of chromosome 21 gene expression. To explore alterations of the GATA1 transcriptome, we used cross-species comparison with genes regulated by GATA1 expression in murine erythroid precursors. Genes repressed after GATA1 induction in the murine system, most notably GATA-2, MYC, and KIT, show increased expression in DS-AMKL, suggesting that GATA1s fail to repress this class of genes. Only a subset of genes that are up-regulated upon GATA1 induction in the murine system show increased expression in DS-AMKL, including GATA1 and BACH1, a probable negative regulator of megakaryocytic differentiation located on chromosome 21. Surprisingly, expression of the chromosome 21 gene RUNX1, a known regulator of megakaryopoiesis, was not elevated in DS-AMKL. Our results identify relevant signatures for distinct AMKL entities and provide insight into gene expression changes associated with these related leukemias.

Chromosomes, Human, Pair 21↗
PubMedDate not supplied

The sentinel within: exploiting the immune system for cancer biomarkers.

The release of proteins from tumors triggers an immune response in cancer patients. These tumor antigens arise from several mechanisms including tumor-specific alterations in protein expression, mutation, folding, degradation, or intracellular localization. Responses to most tumor antigens are rarely observed in healthy individuals, making the response itself a biomarker that betrays the presence of underlying cancer. Antibody immune responses show promise as clinical biomarkers because antibodies have long half-lives in serum, are easy to measure, and are stable in blood samples. However, our understanding of the specificity and the impact of the immune response in early stages of cancer is limited. The immune response to cancer, whether endogenous or driven by vaccines, involves highly specific T lymphocytes (which target tumor-derived peptides bound to self-MHC proteins) and B lymphocytes (which generate antibodies to tumor-derived proteins). T cell target antigens have been identified either by expression cloning from tumor cDNA libraries, or by prediction based on patterns of antigen expression ("reverse immunology"). B cell targets have been similarly identified using the antibodies in patient sera to screen cDNA libraries derived from tumor cell lines. This review focuses on the application of recent advances in proteomics for the identification of tumor antigens. These advances are opening the door for targeted vaccine development, and for using immune response signatures as biomarkers for cancer diagnosis and monitoring.

Antigens, Neoplasm↗