Marie-Laëtitia Thézénas

Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity

Radicals push proteins beyond genes Chemically modifying proteins after their translation can expand their structural and functional roles (see the Perspective by Hofmann and Bode). Two related methods describe how to exploit free radical chemistry to form carbon-carbon bonds between amino acid residues and a selected functional group. Wright et al. added a wide range of functional groups to proteins containing dehydroalanine precursors, with borohydride mediating the radical chemistry. Yang et al. employed a similar approach, using zinc in combination with copper ions. Together, these results will be useful for introducing functionalities and labels to a wide range of proteins. Science, this issue pp. 597 and 623; see also p. 553 Radical chemistry allows for the selective chemical modification of a range of proteins. INTRODUCTION Natural posttranslational modifications (PTMs) to proteins expand the chemical groups available to proteins. The ability to expand posttranslational functional group diversity in an unbounded manner could, in principle, allow exploration and understanding of how these groups modulate biological function. Natural PTMs feature bonds to heteroatoms (non-carbon) made at the γ (Cys Sγ, Thr Oγ, Ser Oγ) or ω (Lys Nω, Tyr Oω) positions of side chains. However, one of the central features of biomolecules is C (sp3)–C (sp3) bond formation. Because all amino acid side chains contain this C–C bond, mastering its construction on proteins could allow free-ranging structural alteration of residues in proteins (both natural and unnatural). RATIONALE In principle, C (sp3)–C (sp3) disconnections at the β,γ C–C bond would allow the chemical installation of a wide range of amino acid functionalities. Traditional two-electron chemistry (using nucleophiles and electrophiles) requires reagents that are often incompatible with biological substrates and/or water. Free radicals can be tolerant of aqueous conditions and unreactive (and thereby compatible) with the majority of functionality present in biomolecules. We therefore reasoned that mild, carbon-centered free radical chemistry would be enabled by matching free-radical reactivity with a suitable, uniquely reactive functional group partner that possesses a chemical affinity for such singly occupied molecular orbitals. The amino acid residue dehydroalanine (Dha) can be readily introduced in a site-selective manner genetically, biosynthetically, or chemically; upon reaction with a suitable radical, Dha would favorably generate a stabilized Cα radical 1. Suitable “quenching” of the central Cα radical intermediate 1 generated after formation of the critical C–C bond would thus allow “chemical mutation” of the side chain. RESULTS A range of precursor halides (R-Hal, Hal = I or Br) allowed the creation of radicals R•. These radicals reacted selectively with Dha in peptides and proteins with excellent site selectivity and regioselectivity (>98% β,γ) and typically with a diastereoselectivity of ~1:1. Combined use of R-Hal with NaBH4 under low-oxygen conditions suppressed competing oxidation and disubstitution side reactions of intermediates 1. This allowed for rapid reactions (typically 30 min) with improved efficiency across a range of representative protein types and scaffolds (all α, α/β folds, all β, receptor, enzyme, antibody). The reactivity of primary, secondary, and tertiary alkyl halides allowed installation of natural, simple hydrophobic residue side chains. Charged or polar protic (e.g., OH, NH) functionality in amino acid side chains was also possible. Even the use of side-chain reagents in unprotected form proved possible, thus highlighting not only exquisite chemoselectivity but also compatibility with common biological functional groups. These transformations enabled the creation of a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated) and labeled (fluorinated, isotopically labeled) side chains, as well as difficult-to-access but important residues in proteins (e.g., methyl-Arg, citrulline, ornithine, methyl-Gln, phospho-Ser). CONCLUSION This approach to chemical editing of amino acid residues, outside of the rigid constraints of the ribosome and enzymatic processing, may prove to be a general technology for accessing diverse, previously unattainable proteins. Posttranslational chemical mutagenesis through C (sp3)–C (sp3) bond-forming radical reactions. Modification in a protein after translation using C–C bond formation allows construction of many side chains, not just the modification of existing natural amino acid residues. t-Leu, tert-leucine; Orn, ornithine; Cit, citrulline. Posttranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. However, the vast diversity of chemically plausible (including unnatural but functionally relevant) side chains is not readily accessible. We describe C (sp3)–C (sp3) bond-forming reactions on proteins under biocompatible conditions, which exploit unusual carbon free-radical chemistry, and use them to form Cβ–Cγ bonds with altered side chains. We demonstrate how these transformations enable a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated), and labeled (fluorinated, isotopically labeled) side chains to be added to a common, readily accessible dehydroalanine precursor in a range of representative protein types and scaffolds. This approach, outside of the rigid constraints of the ribosome and enzymatic processing, may be modified more generally for access to diverse proteins.

CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer

Cells require nucleotides to support DNA replication and repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2′deoxycytidine (5mdC) has been investigated before, it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2′deoxycytidine (5hmdC), 5-formy-2′deoxycytidine (5fdC) and 5-carboxyl-2′deoxycytidine (5cadC). Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogues.

Crystal structure of Porphyromonas gingivalis peptidylarginine deiminase: implications for autoimmunity in rheumatoid arthritis

Background Periodontitis (PD) is a known risk factor for rheumatoid arthritis (RA) and there is increasing evidence that the link between the two diseases is due to citrullination by the unique bacterial peptidylarginine deiminase (PAD) enzyme expressed by periodontal pathogen Pophyromonas gingivalis (PPAD). However, the precise mechanism by which PPAD could generate potentially immunogenic peptides has remained controversial due to lack of information about the structural and catalytic mechanisms of the enzyme. Objectives By solving the 3D structure of PPAD we aim to characterise activity and elucidate potential mechanisms involved in breach of tolerance to citrullinated proteins in RA. Methods PPAD and a catalytically inactive mutant PPADC351A were crystallised and their 3D structures solved. Key residues identified from 3D structures were examined by mutations. Fibrinogen and α-enolase were incubated with PPAD and P. gingivalis arginine gingipain (RgpB) and citrullinated peptides formed were sequenced and quantified by mass spectrometry. Results Here, we solve the crystal structure of a truncated, highly active form of PPAD. We confirm catalysis is mediated by the following residues: Asp130, His236, Asp238, Asn297 and Cys351 and show Arg152 and Arg154 may determine the substrate specificity of PPAD for C-terminal arginines. We demonstrate the formation of 37 C-terminally citrullinated peptides from fibrinogen and 11 from α-enolase following incubation with tPPAD and RgpB. Conclusions PPAD displays an unequivocal specificity for C-terminal arginine residues and readily citrullinates peptides from key RA autoantigens. The formation of these novel citrullinated peptides may be involved in breach of tolerance to citrullinated proteins in RA.

TLR Adaptor Protein MYD88 Mediates Sensitivity to HDAC Inhibitors via a Cytokine-Dependent Mechanism.

Histone deacetylase (HDAC) inhibitors have proven useful therapeutic agents for certain hematologic cancers. However, HDAC inhibition causes diverse cellular outcomes, and identification of cancer-relevant pathways within these outcomes remains unresolved. In this study, we utilized an unbiased loss-of-function screen and identified the Toll-like receptor (TLR) adaptor protein MYD88 as a key regulator of the antiproliferative effects of HDAC inhibition. High expression of MYD88 exhibited increased sensitivity to HDAC inhibitors; conversely, low expression coincided with reduced sensitivity. MYD88-dependent TLR signaling controlled cytokine levels, which then acted via an extracellular mechanism to maintain cell proliferation and sensitize cells to HDAC inhibition. MYD88 activity was directly regulated through lysine acetylation and was deacetylated by HDAC6. MYD88 was a component of a wider acetylation signature in the ABC subgroup of diffuse large B-cell lymphoma, and one of the most frequent mutations in MYD88, L265P, conferred increased cell sensitivity to HDAC inhibitors. Our study defines acetylation of MYD88, which, by regulating TLR-dependent signaling to cytokine genes, influences the antiproliferative effects of HDAC inhibitors. Our results provide a possible explanation for the sensitivity of malignancies of hematologic origin to HDAC inhibitor-based therapy. Cancer Res; 76(23); 6975-87. ©2016 AACR.

Plasma Biomarker Profile Alterations during Variable Blood Storage.

To the Editor: A major confounding factor in the discovery of disease-specific molecular signatures is the variability in handling clinical blood samples. Consequently, novel protein markers of diseases have failed the “iron test” of validation and implementation into clinical practice because, in addition to patient variability, a range of preanalytical parameters contributes to erroneous results. The temperature of storing, transporting, and processing of whole blood following collection is one factor that has not been standardized. Often, samples are transferred from the local laboratories to centralized biobanks in a cooled environment or even on dry ice. Recently, we established a UK biobank to conduct research into the quality of organs donated for transplantation [UK QUOD (Quality in Organ Donation)], collecting donor blood samples from 60 UK transplant centers (www.Quod.org.uk). Taking into consideration the logistical challenges in a clinical setting for sample collection and biobanking, we favored whole blood sample handling and processing at ambient temperature (22 ± 2 °C) before isolation of plasma by centrifugation and subsequent storage at −80 °C. We explored how the proteome and degradome (proteolytic processing of the proteome) may change when whole blood remains at ambient temperature for 30 min, 8 h, 24 h, and 48 h before plasma preparation. We …

Proteo-metabolomics reveals compensation between ischemic and non-injured contralateral kidneys after reperfusion

Ischaemia and reperfusion injury (IRI) is the leading cause of acute kidney injury (AKI), which contributes to high morbidity and mortality rates in a wide range of injuries as well as the development of chronic kidney disease. The cellular and molecular responses of the kidney to IRI are complex and not fully understood. Here, we used an integrated proteomic and metabolomic approach to investigate the effects of IRI on protein abundance and metabolite levels. Rat kidneys were subjected to 45 min of warm ischaemia followed by 4 h and 24 h reperfusion, with contralateral and separate healthy kidneys serving as controls. Kidney tissue proteomics after IRI revealed elevated proteins belonging to the acute phase response, coagulation and complement pathways, and fatty acid (FA) signalling. Metabolic changes were already evident after 4 h reperfusion and showed increased level of glycolysis, lipids and FAs, whilst mitochondrial function and ATP production was impaired after 24 h. This deficit was partially compensated for by the contralateral kidney. Such a metabolic balance counteracts for the developing energy deficit due to reduced mitochondrial function in the injured kidney.

S94 Biological markers of favourable prognosis and successful pleurodesis for malignant pleural effusion

Introduction and objectives Malignant pleural effusion (MPE) is a rapidly rising healthcare burden and critically hampers the patients’ survival and quality of life. Current treatments aim to symptoms’ palliation and talc pleurodesis remains a standard therapeutic modality. There is relatively little high quality research data in prediction of patients’ survival and successful pleurodesis. Therefore prognostic and therapeutic biomarkers are desperately needed. Aim To identify and validate novel prognostic and therapeutic biomarkers in MPE. Methods Clinical data and pleural fluids from MPE patients, prior to treatment have been prospectively collected for TIME2 trial. According to the trial database patients have been classified in two different groups: survival cohort (poor, n = 20/good, n = 14) and treatment outcome cohort (success, n = 15/failure, n = 11). Pleural fluids on enrolment were assessed with mass spectrometry profiling after depletion of the 12 most abundant proteins. Full protein profile analysed with R software and ELISA technique was performed for the validation of the results. Pathway analysis on samples performed with Ingenuity Pathway Analysis software. Results With the use of mass spectrometry we identified 1,154 proteins in the pleural fluid, 167 of which were statistical significant (two tailed T-Test, p < 0.05) between survival groups and 97 of which were statistically significant (two tailed T-Test, p < 0.05) between the pleurodesis groups. Analysis of the data (cross validated by 3 independent core bioinformatic groups) identified 10 survival and 3 pleurodesis biomarkers that were differentially expressed in the favourable prognosis and treatment success group respectively. Exploration of the mass spectrometry data identified pathways that were upregulated on patients with favourable survival that could be used for targeted therapies. Conclusions Based on unique database survival and therapeutic biomarkers were identified that can potentially stratify patients’ management. The results are currently validated on a different retrospective dataset (TIME1 trial) and with a prospective clinical trial (SIMPLE study).