Caroline J. DeHart

Extensive Post-translational Modification of Active and Inactivated Forms of Endogenous p53

The p53 tumor suppressor protein accumulates to very high concentrations in normal human fibroblasts infected by adenovirus type 5 mutants that cannot direct assembly of the viral E1B 55-kDa protein-containing E3 ubiquitin ligase that targets p53 for degradation. Despite high concentrations of nuclear p53, the p53 transcriptional program is not induced in these infected cells. We exploited this system to examine select post-translational modifications (PTMs) present on a transcriptionally inert population of endogenous human p53, as well as on p53 activated in response to etoposide treatment of normal human fibroblasts. These forms of p53 were purified from whole cell lysates by means of immunoaffinity chromatography and SDS-PAGE, and peptides derived from them were subjected to nano-ultra-high-performance LC-MS and MS/MS analyses on a high-resolution accurate-mass MS platform (data available via ProteomeXchange, PXD000464). We identified an unexpectedly large number of PTMs, comprising phosphorylation of Ser and Thr residues, methylation of Arg residues, and acetylation, ubiquitinylation, and methylation of Lys residues—for example, some 150 previously undescribed modifications of p53 isolated from infected cells. These modifications were distributed across all functional domains of both forms of the endogenous human p53 protein, as well as those of an orthologous population of p53 isolated from COS-1 cells. Despite the differences in activity, including greater in vitro sequence-specific DNA binding activity exhibited by p53 isolated from etoposide-treated cells, few differences were observed in the location, nature, or relative frequencies of PTMs on the two populations of human p53. Indeed, the wealth of PTMs that we have identified is consistent with a far greater degree of complex, combinatorial regulation of p53 by PTM than previously anticipated.

The Repression Domain of the E1B 55-Kilodalton Protein Participates in Countering Interferon-Induced Inhibition of Adenovirus Replication

ABSTRACT To begin to investigate the mechanism by which the human adenovirus type 5 E1B 55-kDa protein protects against the antiviral effects of type 1 interferon (IFN) (J. S. Chahal, J. Qi, and S. J. Flint, PLoS Pathog. 8:e1002853, 2012 [doi:10.1371/journal.ppat.1002853]), we examined the effects of precise amino acid substitution in this protein on resistance of viral replication to the cytokine. Only substitution of residues 443 to 448 of E1B for alanine (E1B Sub19) specifically impaired production of progeny virus and resulted in a large defect in viral DNA synthesis in IFN-treated normal human fibroblasts. Untreated or IFN-treated cells infected by this mutant virus (AdEasyE1Sub19) contained much higher steady-state concentrations of IFN-inducible GBP1 and IFIT2 mRNAs than did wild-type-infected cells and of the corresponding newly transcribed pre-mRNAs, isolated exploiting 5′-ethynyluridine labeling and click chemistry. These results indicated that the mutations created by substitution of residues 443 to 448 for alanine (Sub19) impair repression of transcription of IFN-inducible genes, by the E1B, 55-kDa protein, consistent with their location in a segment required for repression of p53-dependent transcription. However, when synthesized alone, the E1B 55-kDa protein inhibited expression of the p53-regulated genes BAX and MDM2 but had no impact whatsoever on induction of IFIT2 and GBP1 expression by IFN. These observations correlate repression of transcription of IFN-inducible genes by the E1B 55-kDa protein with protection against inhibition of viral genome replication and indicate that the E1B 55-kDa protein is not sufficient to establish such transcriptional repression.

Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry

Successful high-throughput characterization of intact proteins from complex biological samples by mass spectrometry requires instrumentation capable of high mass resolving power, mass accuracy, sensitivity, and spectral acquisition rate. These limitations often necessitate the performance of hundreds of LC-MS/MS experiments to obtain reasonable coverage of the targeted proteome, which is still typically limited to molecular weights below 30 kDa. The National High Magnetic Field Laboratory (NHMFL) recently installed a 21 T FT-ICR mass spectrometer, which is part of the NHMFL FT-ICR User Facility and available to all qualified users. Here we demonstrate top-down LC-21 T FT-ICR MS/MS of intact proteins derived from human colorectal cancer cell lysate. We identified a combined total of 684 unique protein entries observed as 3238 unique proteoforms at a 1% false discovery rate, based on rapid, data-dependent acquisition of collision-induced and electron-transfer dissociation tandem mass spectra from just 40 LC-MS/MS experiments. Our identifications included 372 proteoforms with molecular weights over 30 kDa detected at isotopic resolution, which substantially extends the accessible mass range for high-throughput top-down LC-MS/MS.

Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein.

Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087.

Characterization of Methanobactin from Methylosinus sp. LW4

Methanobactins (Mbns) are a growing family of ribosomally produced, post-translationally modified natural products. Characteristic nitrogen-containing heterocycles and neighboring thioamides allow these compounds to bind copper with high affinity. Genome mining has enabled the identification of Mbn operons in bacterial genomes and the prediction of diverse Mbn structures from operon content and precursor peptide sequence. Here we report the characterization of Mbn from Methylosinus (Ms.) species (sp.) LW4. The peptide backbone is distinct from all previously characterized Mbns, and the post-translational modifications correspond precisely to those predicted on the basis of the Ms. sp. LW4 Mbn operon. Thus, prediction based on genome analysis combined with isolation and structural characterization represents a phylogenetic approach to finding diverse Mbns and elucidating their biosynthetic pathways.

High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer

The analysis of intact proteins (top-down strategy) by mass spectrometry has great potential to elucidate proteoform variation, including patterns of post-translational modifications (PTMs), which may not be discernible by analysis of peptides alone (bottom-up approach). To maximize sequence coverage and localization of PTMs, various fragmentation modes have been developed to produce fragment ions from deep within intact proteins. Ultraviolet photodissociation (UVPD) has recently been shown to produce high sequence coverage and PTM retention on a variety of proteins, with increasing evidence of efficacy on a chromatographic time scale. However, utilization of UVPD for high-throughput top-down analysis to date has been limited by bioinformatics. Here we detected 153 proteins and 489 proteoforms using UVPD and 271 proteins and 982 proteoforms using higher energy collisional dissociation (HCD) in a comparative analysis of HeLa whole-cell lysate by qualitative top-down proteomics. Of the total detected proteoforms, 286 overlapped between the UVPD and HCD data sets, with 68% of proteoforms having C scores greater than 40 for UVPD and 63% for HCD. The average sequence coverage (28 ± 20% for UVPD versus 17 ± 8% for HCD, p < 0.0001) was found to be higher for UVPD than HCD and with a trend toward improvement in q value for the UVPD data set. This study demonstrates the complementarity of UVPD and HCD for more extensive protein profiling and proteoform characterization.

The biosynthesis of methanobactin

Using iron to generate a copper ligand Many microbial enzymes are metal-dependent, and the microbe must acquire scarce metals from the environment. Microbes that use methane as a carbon source have a copper-dependent enzyme that oxidizes the methane. Peptides known as methanobactins (Mbns) acquire copper by using a pair of ligands comprising a nitrogen-containing ring and an adjacent thioamide. Kenney et al. describe the biosynthetic machinery that adds the copper-binding groups to a precursor peptide. This involves a complex of two homologs: MbnB, a member of a functionally uncharacterized protein family that includes a diiron cluster, and MbnC, which is even less well characterized. The iron cofactor is required for ligand synthesis. MbnB and MbnC homologs are encoded in many genomes, suggesting that they may have roles beyond Mbn biosynthesis. Science, this issue p. 1411 An enzyme complex uses iron and dioxygen to generate copper-binding ligands in the methanobactin family of natural products. Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.

Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk

Significance The KRAS gene is frequently mutated in human cancer, especially in pancreatic, lung, and colorectal tumors. We developed an intact protein assay for the detection and quantitation of KRAS protein forms (proteoforms), enabling the measurement of how genetically encoded mutations affect posttranslational modifications on the same protein molecule. The modifications found are known to activate KRAS or interfere with membrane attachment, suggesting cellular mechanisms that control downstream KRAS activity. Analysis of wild-type and mutant-specific forms of the KRAS protein with complete molecular specificity is shown to be possible by top-down proteomics and enables future tests of how an individual’s KRAS proteoforms are linked to disease stage and chance of survival. Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.

Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria

Methane-oxidizing microbes catalyze the oxidation of the greenhouse gas methane using the copper-dependent enzyme particulate methane monooxygenase (pMMO). Isolated pMMO exhibits lower activity than whole cells, however, suggesting that additional components may be required. A pMMO homolog, ammonia monooxygenase (AMO), converts ammonia to hydroxylamine in ammonia-oxidizing bacteria (AOB) which produce another potent greenhouse gas, nitrous oxide. Here we show that PmoD, a protein encoded within many pmo operons that is homologous to the AmoD proteins encoded within AOB amo operons, forms a copper center that exhibits the features of a well-defined CuA site using a previously unobserved ligand set derived from a cupredoxin homodimer. PmoD is critical for copper-dependent growth on methane, and genetic analyses strongly support a role directly related to pMMO and AMO. These findings identify a copper-binding protein that may represent a missing link in the function of enzymes critical to the global carbon and nitrogen cycles.Methane- and ammonia-oxidizing bacteria use the integral membrane, copper-dependent enzymes particulate methane monooxygenase (pMMO) and ammonia monooxygenase (AMO) to oxidize methane and ammonia. Here the authors structurally characterize the copper-binding protein PmoD, which contains an unusual CuA site and their genetic analyses strongly support a pMMO and AMO related function of PmoD.

The unique biomineralization transcriptome and proteome of Lytechinus variegatus teeth

ABSTRACT Background: Matrix-regulated biomineralization involves the specific nucleation and growth of mineral phases within or upon preformed structured organic matrices. We hypothesized that there might be a general mechanism whereby anionic, phosphorylated mineral ion-binding proteins assist in specifically locating the mineral ions with respect to the mineralizing structural organic matrix. Here we extended these studies to invertebrate mineralization in Lytechinus variegatus (Lv) teeth. Materials and Methods: The tooth proteins were extracted and the phosphoproteins occluded in the mineral were enriched by passage through a ProQ Diamond phosphoprotein enrichment column, and subjected to MS/MS analysis. A Lv RNA-seq derived transcriptome database was generated. The MS/MS data found 25 proteins previously classified as “Predicted uncharacterized proteins” and many of the spicule matrix proteins. As these 25 proteins were also identified with the transcriptome analysis, and were thus no longer “hypothetical” but real proteins in the Lv tooth. Each protein was analyzed for the presence of a signal peptide, an acidic pI≤4, and the ability to be phosphorylated. Results: Four new Lv tooth specific Pro-Ala-rich proteins were found, representing a new class of proteins. Conclusion: The tooth is different from the spicules and other urchin skeletal elements in that only the tooth contains both “high” and “very high” magnesium calcite, [Ca(1–X) Mg(X) CO3], where X is the mole fraction of Mg. We speculate that our newly discovered proline-alanine rich proteins, also containing sequences of acidic amino acids, may be involved in the formation of high magnesium and very high magnesium calcite.