Publications by authors named "Eranthie Weerapana"

100 Publications

Ferlins and TgDOC2 in Microneme, Rhoptry and Dense Granule Secretion.

Life (Basel) 2021 Mar 9;11(3). Epub 2021 Mar 9.

Department of Biology, Boston College, Chestnut Hill, MA 02467, USA.

The host cell invasion process of apicomplexan parasites like is facilitated by sequential exocytosis of the microneme, rhoptry and dense granule organelles. Exocytosis is facilitated by a double C2 domain (DOC2) protein family. This class of C2 domains is derived from an ancestral calcium (Ca) binding archetype, although this feature is optional in extant C2 domains. DOC2 domains provide combinatorial power to the C2 domain, which is further enhanced in ferlins that harbor 5-7 C2 domains. Ca conditionally engages the C2 domain with lipids, membranes, and/or proteins to facilitating vesicular trafficking and membrane fusion. The widely conserved ferlins 1 (FER1) and 2 (FER2) are responsible for microneme and rhoptry exocytosis, respectively, whereas an unconventional TgDOC2 is essential for microneme exocytosis. The general role of ferlins in endolysosmal pathways is consistent with the repurposed apicomplexan endosomal pathways in lineage specific secretory organelles. Ferlins can facilitate membrane fusion without SNAREs, again pertinent to the Apicomplexa. How temporal raises in Ca combined with spatiotemporally available membrane lipids and post-translational modifications mesh to facilitate sequential exocytosis events is discussed. In addition, new data on cross-talk between secretion events together with the identification of a new microneme protein, MIC21, is presented.
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http://dx.doi.org/10.3390/life11030217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999867PMC
March 2021

Gibberellin JRA-003: A Selective Inhibitor of Nuclear Translocation of IKKα.

ACS Med Chem Lett 2020 Oct 21;11(10):1913-1918. Epub 2020 May 21.

Department of Chemistry, Willard-Henry-Dow Laboratory, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States.

The small molecule gibberellin JRA-003 was identified as an inhibitor of the NF-kB (nuclear kappa-light-chain-enhancer of activated B cells) pathway. Here we find that JRA-003 binds to and significantly inhibits the nuclear translocation of pathway-activating kinases IKKα (IκB kinase alpha) and IKKβ (IκB kinase beta). Analogs of JRA-003 were synthesized and NF-κB-inhibiting gibberellins were found to be cytotoxic in cancer-derived cell lines (HS 578T, HCC 1599, RC-K8, Sud-HL4, CA 46, and NCIH 4466). Not only was JRA-003 identified as the most potent synthetic gibberellin against cancer-derived cell lines, it displayed no cytotoxicity in cells derived from noncancerous sources (HEK 293T, HS 578BST, HS 888Lu, HS 895Sk, HUVEC). This selectivity suggests a promising approach for the development of new therapeutics.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549252PMC
October 2020

Chemical Tools in Biological Discovery.

Cell Chem Biol 2020 08;27(8):889-890

Associate Editor, Cell Chemical Biology.

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http://dx.doi.org/10.1016/j.chembiol.2020.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439815PMC
August 2020

Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite.

J Biol Chem 2020 09 19;295(39):13410-13418. Epub 2020 Aug 19.

Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA. Electronic address:

An important context in which metabolism influences tumorigenesis is the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand the potential for metabolites to influence posttranslational modifications important to tumorigenesis and cancer cell growth, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Using a general reactivity probe, we generated a data set of proteomic cysteine residues sensitive to the reduction in fumarate levels caused by genetic reintroduction of active FH into HLRCC cell lines. This revealed a broad up-regulation of cysteine reactivity upon FH rescue, which evidence suggests is caused by an approximately equal proportion of transcriptional and posttranslational modification-mediated regulation. Gene ontology analysis highlighted several new targets and pathways potentially modulated by FH mutation. Comparison of the new data set with prior studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. This is consistent with emerging studies indicating the existence of cell- and tissue-specific cysteine-omes, further emphasizing the need for characterization of diverse models. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation and a foundation for future efforts to exploit this knowledge for cancer therapy.
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http://dx.doi.org/10.1074/jbc.AC120.014993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521642PMC
September 2020

Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine.

ACS Chem Biol 2020 06 5;15(6):1535-1540. Epub 2020 May 5.

Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, United States.

Selenoproteins contain the amino acid selenocysteine (Sec) and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a noncanonical translation mechanism requiring suppression of the UGA stop codon and a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element and selenium availability. An engineered orthogonal . leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged Sec (DMNB-Sec) at the UAG amber stop codon. DMNB-Sec is successfully incorporated into GFP and uncaged by irradiation of living cells. Furthermore, DMNB-Sec is used to generate the native selenoprotein methionine-R-sulfoxide reductase B1 (MsrB1). Importantly, MsrB1 is shown to be catalytically active after uncaging, constituting the first use of genetic code expansion to generate a functional selenoprotein in mammalian systems. The ability to site-specifically introduce Sec directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.
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http://dx.doi.org/10.1021/acschembio.0c00147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372505PMC
June 2020

Characterization of Serine Hydrolases Across Clinical Isolates of Commensal Skin Bacteria Using Activity-Based Protein Profiling.

ACS Infect Dis 2020 05 27;6(5):930-938. Epub 2020 Apr 27.

Department of Bioengineering and ChEM-H, Stanford University, Stanford, California 94305, United States.

The bacterial genus comprises diverse species that colonize the skin as commensals but can also cause infection. Previous work identified a family of serine hydrolases termed fluorophoshonate-binding hydrolases (Fphs) in the pathogenic bacteria , one of which, FphB, functions as a virulence factor. Using a combination of bioinformatics and activity-based protein profiling (ABPP), we identify homologues of these enzymes in the related commensal bacteria . Two of the Fph enzymes were not identified in . Using ABPP, we identified several candidate hydrolases that were not previously identified in that may be functionally related to the Fphs. Interestingly, the activity of the Fphs vary across clinical isolates of . Biochemical characterization of the FphB homologue in (SeFphB) suggests it is a functional homologue of FphB in , but our preliminary studies suggest it may not have a role in colonization . This potential difference in biological function between the Fphs of closely related staphylococcal species may provide mechanisms for specific inhibition of infection without perturbing commensal communities of related bacteria.
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http://dx.doi.org/10.1021/acsinfecdis.0c00095DOI Listing
May 2020

Genetically encoded protein sulfation in mammalian cells.

Nat Chem Biol 2020 04 16;16(4):379-382. Epub 2020 Mar 16.

Department of Chemistry, Boston College, Chestnut Hill, MA, USA.

Tyrosine sulfation is an important post-translational modification found in higher eukaryotes. Here we report an engineered tyrosyl-tRNA synthetase/tRNA pair that co-translationally incorporates O-sulfotyrosine in response to UAG codons in Escherichia coli and mammalian cells. This platform enables recombinant expression of eukaryotic proteins homogeneously sulfated at chosen sites, which was demonstrated by expressing human heparin cofactor II in mammalian cells in different states of sulfation.
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http://dx.doi.org/10.1038/s41589-020-0493-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564891PMC
April 2020

The Antimalarial Natural Product Salinipostin A Identifies Essential α/β Serine Hydrolases Involved in Lipid Metabolism in P. falciparum Parasites.

Cell Chem Biol 2020 02 23;27(2):143-157.e5. Epub 2020 Jan 23.

Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/β serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.
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http://dx.doi.org/10.1016/j.chembiol.2020.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027986PMC
February 2020

Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum.

ACS Chem Biol 2020 02 15;15(2):543-553. Epub 2020 Jan 15.

Department of Chemistry , Boston College , Chestnut Hill , Massachusetts 02467 , United States.

The endoplasmic reticulum (ER) is the initial site of biogenesis of secretory pathway proteins, including proteins localized to the ER, Golgi, lysosomes, intracellular vesicles, plasma membrane, and extracellular compartments. Proteins within the secretory pathway contain a high abundance of disulfide bonds to protect against the oxidative extracellular environment. These disulfide bonds are typically formed within the ER by a variety of oxidoreductases, including members of the protein disulfide isomerase (PDI) family. Here, we establish chemoproteomic platforms to identify oxidized and reduced cysteine residues within the ER. Subcellular fractionation methods were utilized to enrich for the ER and significantly enhance the coverage of ER-localized cysteine residues. Reactive-cysteine profiling ranked ∼900 secretory pathway cysteines by reactivity with an iodoacetamide-alkyne probe, revealing functional cysteines annotated to participate in disulfide bonds, or palmitoylation sites within proteins. Through application of a variation of the OxICAT protocol for quantifying cysteine oxidation, the percentages of oxidation for each of ∼700 ER-localized cysteines were calculated. Lastly, perturbation of ER function, through chemical induction of ER stress, was used to investigate the effect of initiation of the unfolded protein response (UPR) on ER-localized cysteine oxidation. Together, these studies establish a platform for identifying reactive and functional cysteine residues on proteins within the secretory pathway as well as for interrogating the effects of diverse cellular stresses on ER-localized cysteine oxidation.
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http://dx.doi.org/10.1021/acschembio.9b01014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650206PMC
February 2020

Reciprocal regulation of Th2 and Th17 cells by PAD2-mediated citrullination.

JCI Insight 2019 11 14;4(22). Epub 2019 Nov 14.

Division of Rheumatology, Inflammation, and Immunity, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.

Dysregulated citrullination, a unique form of posttranslational modification catalyzed by the peptidylarginine deiminases (PADs), has been observed in several human diseases, including rheumatoid arthritis. However, the physiological roles of PADs in the immune system are still poorly understood. Here, we report that global inhibition of citrullination enhances the differentiation of type 2 helper T (Th2) cells but attenuates the differentiation of Th17 cells, thereby increasing the susceptibility to allergic airway inflammation. This effect on Th cells is due to inhibition of PAD2 but not PAD4. Mechanistically, PAD2 directly citrullinates GATA3 and RORγt, 2 key transcription factors determining the fate of differentiating Th cells. Citrullination of R330 of GATA3 weakens its DNA binding ability, whereas citrullination of 4 arginine residues of RORγt strengthens its DNA binding. Finally, PAD2-deficient mice also display altered Th2/Th17 immune response and heightened sensitivity to allergic airway inflammation. Thus, our data highlight the potential and caveat of PAD2 as a therapeutic target of Th cell-mediated diseases.
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http://dx.doi.org/10.1172/jci.insight.129687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948856PMC
November 2019

Chemical Biology Approaches to Interrogate the Selenoproteome.

Acc Chem Res 2019 10 16;52(10):2832-2840. Epub 2019 Sep 16.

Department of Chemistry , Boston College , Chestnut Hill , Massachusetts 02467 , United States.

Selenoproteins are the family of proteins that contain the amino acid selenocysteine. Many selenoproteins, including glutathione peroxidases and thioredoxin reductases, play a role in maintaining cellular redox homeostasis. There are a number of examples of homologues of selenoproteins that utilize cysteine residues, raising the question of why selenocysteines are utilized. One hypothesis is that incorporation of selenocysteine protects against irreversible overoxidation, typical of cysteine-containing homologues under high oxidative stress. Studies of selenocysteine function are hampered by challenges both in detection and in recombinant expression of selenoproteins. In fact, about half of the 25 known human selenoproteins remain uncharacterized. Historically, selenoproteins were first detected via labeling with radioactive Se or by use of inductively coupled plasma-mass spectrometry to monitor nonradioactive selenium. More recently, tandem mass-spectrometry techniques have been developed to detect selenocysteine-containing peptides. For example, the isotopic distribution of selenium has been used as a unique signature to identify selenium-containing peptides from unenriched proteome samples. Additionally, selenocysteine-containing proteins and peptides were selectively enriched using thiol-reactive electrophiles by exploiting the increased reactivity of selenols relative to thiols, especially under low pH conditions. Importantly, the reactivity-based enrichment of selenoproteins can differentiate between oxidized and reduced selenoproteins, providing insight into the activity state. These mass spectrometry-based selenoprotein detection approaches have enabled (1) production of selenoproteome expression atlases, (2) identification of aging-associated changes in selenoprotein expression, (3) characterization of selenocysteine reactivity across the selenoprotein family, and (4) interrogation of selenoprotein targets of small-molecule drugs. Further investigations of selenoprotein function would benefit from recombinant expression of selenoproteins. However, the endogenous mechanism of selenoprotein production makes recombinant expression challenging. Primarily, selenocysteine is biosynthesized on its own tRNA, is dependent on multiple enzymatic steps, and is highly sensitive to selenium concentrations. Furthermore, selenocysteine is encoded by the stop codon UGA, and suppression of that stop codon requires a selenocysteine insertion sequence element in the selenoprotein mRNA. In order to circumvent the low efficiency of the endogenous machinery, selenoproteins have been produced through native chemical ligation and expressed protein ligation. Attempts have also been made to engineer the endogenous machinery for increased efficiency, including recoding the selenocysteine codon, and engineering the tRNA and the selenocysteine insertion sequence element. Alternatively, genetic code expansion can be used to generate selenoproteins. This approach allows for selenoprotein production directly within its native cellular environment, while bypassing the endogenous selenocysteine incorporation machinery. Furthermore, by incorporating a caged selenocysteine by genetic code expansion, selenoprotein activity can be spatially and temporally controlled. Genetic code expansion has allowed for the expression and uncaging of human selenoproteins in and more recently in mammalian cells. Together, advances in selenoprotein detection and expression should enable a better understanding of selenoprotein function and provide insight into the necessity for selenocysteine production.
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http://dx.doi.org/10.1021/acs.accounts.9b00379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953406PMC
October 2019

The apical annuli of Toxoplasma gondii are composed of coiled-coil and signalling proteins embedded in the inner membrane complex sutures.

Cell Microbiol 2020 01 10;22(1):e13112. Epub 2019 Sep 10.

Department of Biology, Boston College, Chestnut Hill, Massachusetts.

The apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2, and an apical annuli methyltransferase. Moreover, inner membrane complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy identified a concentric organisation comprising four rings with diameters ranging from 200 to 400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggests that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed that the AAPs are conserved narrowly in coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signalling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled-coil and signalling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.
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http://dx.doi.org/10.1111/cmi.13112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925623PMC
January 2020

A cysteinyl-tRNA synthetase variant confers resistance against selenite toxicity and decreases selenocysteine misincorporation.

J Biol Chem 2019 08 11;294(34):12855-12865. Epub 2019 Jul 11.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511

Selenocysteine (Sec) is the 21st genetically encoded amino acid in organisms across all domains of life. Although structurally similar to cysteine (Cys), the Sec selenol group has unique properties that are attractive for protein engineering and biotechnology applications. Production of designer proteins with Sec (selenoproteins) at desired positions is now possible with engineered translation systems in However, obtaining pure selenoproteins at high yields is limited by the accumulation of free Sec in cells, causing undesired incorporation of Sec at Cys codons due to the inability of cysteinyl-tRNA synthetase (CysRS) to discriminate against Sec. Sec misincorporation is toxic to cells and causes protein aggregation in yeast. To overcome this limitation, here we investigated a CysRS from the selenium accumulator plant that is reported to reject Sec Sequence analysis revealed a rare His → Asn variation adjacent to the CysRS catalytic pocket. Introducing this variation into and CysRS increased resistance to the toxic effects of selenite and selenomethionine (SeMet), respectively. Although the CysRS variant could still use Sec as a substrate , we observed a reduction in the frequency of Sec misincorporation at Cys codons We surmise that the His → Asn variation can be introduced into any CysRS to provide a fitness advantage for strains burdened by Sec misincorporation and selenium toxicity. Our results also support the notion that the CysRS variant provides higher specificity for Cys as a mechanism for plants to grow in selenium-rich soils.
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http://dx.doi.org/10.1074/jbc.RA119.008219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709638PMC
August 2019

Halogen Bonding Increases the Potency and Isozyme Selectivity of Protein Arginine Deiminase 1 Inhibitors.

Angew Chem Int Ed Engl 2019 09 1;58(36):12476-12480. Epub 2019 Aug 1.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation street, Worcester, MA, 01605, USA.

Protein arginine deiminases (PADs) hydrolyze the side chain of arginine to form citrulline. Aberrant PAD activity is associated with rheumatoid arthritis, multiple sclerosis, lupus, and certain cancers. These pathologies established the PADs as therapeutic targets and multiple PAD inhibitors are known. Herein, we describe the first highly potent PAD1-selective inhibitors (1 and 19). Detailed structure-activity relationships indicate that their potency and selectivity is due to the formation of a halogen bond with PAD1. Importantly, these inhibitors inhibit histone H3 citrullination in HEK293TPAD1 cells and mouse zygotes with excellent potency. Based on this scaffold, we also developed a PAD1-selective activity-based probe that shows remarkable cellular efficacy and proteome selectivity. Based on their potency and selectivity we expect that 1 and 19 will be widely used chemical tools to understand PAD1 biology.
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http://dx.doi.org/10.1002/anie.201906334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713606PMC
September 2019

Plasma Peptidylarginine Deiminase IV Promotes VWF-Platelet String Formation and Accelerates Thrombosis After Vessel Injury.

Circ Res 2019 08 28;125(5):507-519. Epub 2019 Jun 28.

From the Program in Cellular and Molecular Medicine (N.S., D.M.M., K.M., D.C., C.S., D.D.W.), Boston Children's Hospital, MA.

Rationale: PAD4 (peptidylarginine deiminase type IV), an enzyme essential for neutrophil extracellular trap formation (NETosis), is released together with neutrophil extracellular traps into the extracellular milieu. It citrullinates histones and holds the potential to citrullinate other protein targets. While NETosis is implicated in thrombosis, the impact of the released PAD4 is unknown.

Objective: This study tests the hypothesis that extracellular PAD4, released during inflammatory responses, citrullinates plasma proteins, thus affecting thrombus formation.

Methods And Results: Here, we show that injection of r-huPAD4 in vivo induces the formation of VWF (von Willebrand factor)-platelet strings in mesenteric venules and that this is dependent on PAD4 enzymatic activity. VWF-platelet strings are naturally cleaved by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 motif-13). We detected a reduction of endogenous ADAMTS13 activity in the plasma of wild-type mice injected with r-huPAD4. Using mass spectrometry and in vitro studies, we found that r-huPAD4 citrullinates ADAMTS13 on specific arginine residues and that this modification dramatically inhibits ADAMTS13 enzymatic activity. Elevated citrullination of ADAMTS13 was observed in plasma samples of patients with sepsis or noninfected patients who were elderly (eg, age >65 years) and had underlying comorbidities (eg, diabetes mellitus and hypertension) as compared with healthy donors. This shows that ADAMTS13 is citrullinated in vivo. VWF-platelet strings that form on venules of Adamts13 mice were immediately cleared after injection of r-huADAMTS13, while they persisted in vessels of mice injected with citrullinated r-huADAMTS13. Next, we assessed the effect of extracellular PAD4 on platelet-plug formation after ferric chloride-induced injury of mesenteric venules. Administration of r-huPAD4 decreased time to vessel occlusion and significantly reduced thrombus embolization.

Conclusions: Our data indicate that PAD4 in circulation reduces VWF-platelet string clearance and accelerates the formation of a stable platelet plug after vessel injury. We propose that this effect is, at least in part, due to ADAMTS13 inhibition.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.314571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697196PMC
August 2019

Calcium Regulates the Nuclear Localization of Protein Arginine Deiminase 2.

Biochemistry 2019 07 27;58(27):3042-3056. Epub 2019 Jun 27.

Department of Biochemistry and Pharmacology , University of Massachusetts Medical School , Worcester , Massachusetts 01605 , United States.

Protein arginine deiminases (PADs) are calcium-dependent enzymes that mediate the post-translational conversion of arginine into citrulline. Dysregulated PAD activity is associated with numerous autoimmune disorders and cancers. In breast cancer, PAD2 citrullinates histone H3R26 and activates the transcription of estrogen receptor target genes. However, PAD2 lacks a canonical nuclear localization sequence, and it is unclear how this enzyme is transported into the nucleus. Here, we show for the first time that PAD2 translocates into the nucleus in response to calcium signaling. Using BioID2, a proximity-dependent biotinylation method for identifying interacting proteins, we found that PAD2 preferentially associates with ANXA5 in the cytoplasm. Binding of calcium to PAD2 weakens this cytoplasmic interaction, which generates a pool of calcium-bound PAD2 that can interact with Ran. We hypothesize that this latter interaction promotes the translocation of PAD2 into the nucleus. These findings highlight a critical role for ANXA5 in regulating PAD2 and identify an unusual mechanism whereby proteins translocate between the cytosol and nucleus.
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http://dx.doi.org/10.1021/acs.biochem.9b00225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691507PMC
July 2019

Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells.

J Biol Chem 2019 08 18;294(32):12077-12090. Epub 2019 Jun 18.

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109. Electronic address:

Unlike most other tissues, the colon epithelium is exposed to high levels of HS derived from gut microbial metabolism. HS is a signaling molecule that modulates various physiological effects. It is also a respiratory toxin that inhibits complex IV in the electron transfer chain (ETC). Colon epithelial cells are adapted to high environmental HS exposure as they harbor an efficient mitochondrial HS oxidation pathway, which is dedicated to its disposal. Herein, we report that the sulfide oxidation pathway enzymes are apically localized in human colonic crypts at the host-microbiome interface, but that the normal apical-to-crypt gradient is lost in colorectal cancer epithelium. We found that sulfide quinone oxidoreductase (SQR), which catalyzes the committing step in the mitochondrial sulfide oxidation pathway and couples to complex III, is a critical respiratory shield against HS poisoning. HS at concentrations ≤20 μm stimulated the oxygen consumption rate in colon epithelial cells, but, when SQR expression was ablated, HS concentrations as low as 5 μm poisoned cells. Mitochondrial HS oxidation altered cellular bioenergetics, inducing a reductive shift in the NAD/NADH redox couple. The consequent electron acceptor insufficiency caused uridine and aspartate deficiency and enhanced glutamine-dependent reductive carboxylation. The metabolomic signature of this HS-induced stress response mapped, in part, to redox-sensitive nodes in central carbon metabolism. Colorectal cancer tissues and cell lines appeared to counter the growth-restricting effects of HS by overexpressing sulfide oxidation pathway enzymes. Our findings reveal an alternative mechanism for HS signaling, arising from alterations in mitochondrial bioenergetics that drive metabolic reprogramming.
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http://dx.doi.org/10.1074/jbc.RA119.009442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690701PMC
August 2019

Triazine Probes Target Ascorbate Peroxidases in Plants.

Plant Physiol 2019 08 28;180(4):1848-1859. Epub 2019 May 28.

Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom

Though they are rare in nature, anthropogenic 1,3,5-triazines have been used in herbicides as chemically stable scaffolds. Here, we show that small 1,3,5-triazines selectively target ascorbate peroxidases (APXs) in Arabidopsis (), tomato (), rice (), maize (), liverwort (), and other plant species. The alkyne-tagged 2-chloro-4-methyl-1,3,5-triazine probe KSC-3 selectively binds APX enzymes, both in crude extracts and in living cells. KSC-3 blocks APX activity, thereby reducing photosynthetic activity under moderate light stress, even in mutant plants. This suggests that APX enzymes in addition to APX1 protect the photosystem against reactive oxygen species. Profiling APX1 with KCS-3 revealed that the catabolic products of atrazine (a 1,3,5-triazine herbicide), which are common soil pollutants, also target APX1. Thus, KSC-3 is a powerful chemical probe to study APX enzymes in the plant kingdom.
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http://dx.doi.org/10.1104/pp.19.00481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670103PMC
August 2019

Diverse compounds from pleuromutilin lead to a thioredoxin inhibitor and inducer of ferroptosis.

Nat Chem 2019 06 13;11(6):521-532. Epub 2019 May 13.

Department of Chemistry, Roger Adams Laboratory, University of Illinois, Urbana, IL, USA.

The chemical diversification of natural products provides a robust and general method for the creation of stereochemically rich and structurally diverse small molecules. The resulting compounds have physicochemical traits different from those in most screening collections, and as such are an excellent source for biological discovery. Herein, we subject the diterpene natural product pleuromutilin to reaction sequences focused on creating ring system diversity in few synthetic steps. This effort resulted in a collection of compounds with previously unreported ring systems, providing a novel set of structurally diverse and highly complex compounds suitable for screening in a variety of different settings. Biological evaluation identified the novel compound ferroptocide, a small molecule that rapidly and robustly induces ferroptotic death of cancer cells. Target identification efforts and CRISPR knockout studies reveal that ferroptocide is an inhibitor of thioredoxin, a key component of the antioxidant system in the cell. Ferroptocide positively modulates the immune system in a murine model of breast cancer and will be a useful tool to study the utility of pro-ferroptotic agents for treatment of cancer.
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http://dx.doi.org/10.1038/s41557-019-0261-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639018PMC
June 2019

Interrogation of Functional Mitochondrial Cysteine Residues by Quantitative Mass Spectrometry.

Methods Mol Biol 2019 ;1967:211-227

Department of Chemistry, Boston College, Chestnut Hill, MA, USA.

Mitochondria are cellular sites of diverse redox biology, including ROS production, iron-sulfur biogenesis, and secondary metabolism, which all rely on proteogenic reactive cysteine residues. Mass spectrometry-based proteomic methods to monitor the reactivity and functionality of cysteine residues across complex proteomes have greatly expanded over the past decade. Here we describe a mitochondrial isolation procedure coupled with cysteine-reactive IA labeling that affords identification and characterization of functional mitochondrial cysteine residues that were heretofore inaccessible to whole-cell proteomic analysis.
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http://dx.doi.org/10.1007/978-1-4939-9187-7_13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953394PMC
November 2019

Interrogation of Functional Mitochondrial Cysteine Residues by Quantitative Mass Spectrometry.

Methods Mol Biol 2019 ;1967:211-227

Department of Chemistry, Boston College, Chestnut Hill, MA, USA.

Mitochondria are cellular sites of diverse redox biology, including ROS production, iron-sulfur biogenesis, and secondary metabolism, which all rely on proteogenic reactive cysteine residues. Mass spectrometry-based proteomic methods to monitor the reactivity and functionality of cysteine residues across complex proteomes have greatly expanded over the past decade. Here we describe a mitochondrial isolation procedure coupled with cysteine-reactive IA labeling that affords identification and characterization of functional mitochondrial cysteine residues that were heretofore inaccessible to whole-cell proteomic analysis.
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http://dx.doi.org/10.1007/978-1-4939-9187-7_13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953394PMC
November 2019

Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct Sites.

J Am Chem Soc 2019 04 8;141(15):6204-6212. Epub 2019 Apr 8.

Department of Chemistry , Boston College , 2609 Beacon Street, 246B Merkert Chemistry Center , Chestnut Hill , Massachusetts 02467 , United States.

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into a protein is an emerging technology with tremendous potential. It relies on mutually orthogonal engineered aminoacyl-tRNA synthetase/tRNA pairs that suppress different nonsense/frameshift codons. So far, up to two distinct ncAAs have been incorporated into proteins expressed in E. coli, using archaea-derived tyrosyl and pyrrolysyl pairs. Here we report that the E. coli derived tryptophanyl pair can be combined with the archaeal tyrosyl or the pyrrolysyl pair in ATMW1 E. coli to incorporate two different ncAAs into one protein with high fidelity and efficiency. By combining all three orthogonal pairs, we further demonstrate simultaneous site-specific incorporation of three different ncAAs into one protein. To use this technology for chemoselectively labeling proteins with multiple distinct entities at predefined sites, we also sought to identify different bioconjugation handles that can be coincorporated into proteins as ncAA-side chains and subsequently functionalized through mutually compatible labeling chemistries. To this end, we show that the recently developed chemoselective rapid azo-coupling reaction (CRACR) directed to 5-hydroxytryptophan (5HTP) is compatible with strain-promoted azide-alkyne cycloaddition (SPAAC) targeted to p-azidophenylalanine (pAzF) and strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) targeted to cyclopropene-lysine (CpK) for rapid, catalyst-free protein labeling at multiple sites. Combining these mutually orthogonal nonsense suppression systems and the mutually compatible bioconjugation handles they incorporate, we demonstrate site-specific labeling of recombinantly expressed proteins at up to three distinct sites.
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http://dx.doi.org/10.1021/jacs.8b12954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6500092PMC
April 2019

Reactive-cysteine profiling for drug discovery.

Curr Opin Chem Biol 2019 06 18;50:29-36. Epub 2019 Mar 18.

Department of Chemistry, Boston College, Chestnut Hill, MA 02467, United States. Electronic address:

The recognition that only a small percentage of known human gene products are druggable using traditional modes of non-covalent ligand design, has led to a resurgence in targeted covalent inhibitors. Covalent inhibitors offer advantages over non-covalent inhibitors in engaging otherwise challenging targets. Reactive cysteine residues on proteins are a common target for covalent inhibitors, whereby the high nucleophilicity of the cysteine thiol under physiological conditions provides an ideal anchoring site for electrophilic small molecules. A chemical-proteomic platform, termed isoTOP-ABPP, allows for profiling cysteine reactivity in complex proteomes and is one of many techniques that can aid in two aspects of the covalent-inhibitor development process: (1) to identify functional cysteines that lead to modulation of protein activity through covalent modification; and, (2) to determine cellular targets and evaluate promiscuity of electrophilic fragments, small molecules, and natural products. Herein, we discuss recent advances in isoTOP-ABPP and potential applications of this technology in the drug-discovery pipeline.
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http://dx.doi.org/10.1016/j.cbpa.2019.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584045PMC
June 2019

A chemoproteomic portrait of the oncometabolite fumarate.

Nat Chem Biol 2019 04 4;15(4):391-400. Epub 2019 Feb 4.

Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MA, USA.

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.
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http://dx.doi.org/10.1038/s41589-018-0217-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430658PMC
April 2019

Diarylcarbonates are a new class of deubiquitinating enzyme inhibitor.

Bioorg Med Chem Lett 2019 01 28;29(2):204-211. Epub 2018 Nov 28.

Department of Biology, Brandeis University, 415 South Street, Waltham, MA 02453, USA; Department of Chemistry(3), Brandeis University, 415 South Street, Waltham, MA 02453, USA. Electronic address:

Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be similarly valuable. The currently available promiscuous DUB inhibitors are highly reactive electrophilic compounds that can crosslink proteins. Herein we introduce diarylcarbonate esters as a novel class of promiscuous DUB inhibitors that do not have the liabilities associated with the previously reported compounds. Diarylcarbonates stabilize the high molecular weight ubiquitin pools in cells and lysates. They also elicit cellular phenotypes associated with DUB inhibition, demonstrating their utility in ubiquitin discovery. Diarylcarbonates may also be a useful scaffold for the development of specific DUB inhibitors.
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http://dx.doi.org/10.1016/j.bmcl.2018.11.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467287PMC
January 2019

Cysteine reactivity across the subcellular universe.

Curr Opin Chem Biol 2019 02 30;48:96-105. Epub 2018 Nov 30.

Department of Chemistry, Boston College, Chestnut Hill, MA 02467, United States. Electronic address:

Cysteine residues are concentrated at key functional sites within proteins, performing diverse roles in metal binding, catalysis, and redox chemistry. Chemoproteomic platforms to interrogate the reactive cysteinome have developed significantly over the past 10 years, resulting in a greater understanding of cysteine functionality, modification, and druggability. Recently, chemoproteomic methods to examine reactive cysteine residues from specific subcellular organelles have provided significantly improved proteome coverage and highlights the unique functionalities of cysteine residues mediated by cellular localization. Here, the diverse physicochemical properties of the mammalian subcellular organelles are explored in the context of their effects on cysteine reactivity. The unique functions of cysteine residues found in the mitochondria and endoplasmic reticulum are highlighted, together with an overview into chemoproteomic platforms employed to investigate cysteine reactivity in subcellular organelles.
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http://dx.doi.org/10.1016/j.cbpa.2018.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382561PMC
February 2019

Selenium-Encoded Isotopic Signature Targeted Profiling.

ACS Cent Sci 2018 Aug 16;4(8):960-970. Epub 2018 Jul 16.

Synthetic and Functional Biomolecules Center; Beijing National Laboratory for Molecular Sciences; Key Laboratory of Bioorganic Chemistry and Molecular Engineering of the Ministry of Education; College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.

Selenium (Se), as an essential trace element, plays crucial roles in many organisms including humans. The biological functions of selenium are mainly mediated by selenoproteins, a unique class of selenium-containing proteins in which selenium is inserted in the form of selenocysteine. Due to their low abundance and uneven tissue distribution, detection of selenoproteins within proteomes is very challenging, and therefore functional studies of these proteins are limited. In this study, we developed a computational method, named as selenium-encoded isotopic signature targeted profiling (SESTAR), which utilizes the distinct natural isotopic distribution of selenium to assist detection of trace selenium-containing signals from shotgun-proteomic data. SESTAR can detect femtomole quantities of synthetic selenopeptides in a benchmark test and dramatically improved detection of native selenoproteins from tissue proteomes in a targeted profiling mode. By applying SESTAR to screen publicly available datasets from Human Proteome Map, we provide a comprehensive picture of selenoprotein distributions in human primary hematopoietic cells and tissues. We further demonstrated that SESTAR can aid chemical-proteomic strategies to identify additional selenoprotein targets of RSL3, a canonical inducer of cell ferroptosis. We believe SESTAR not only serves as a powerful tool for global profiling of native selenoproteomes, but can also work seamlessly with chemical-proteomic profiling strategies to enhance identification of target proteins, post-translational modifications, or protein-protein interactions.
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http://dx.doi.org/10.1021/acscentsci.8b00112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107865PMC
August 2018

NonA and CPX Link the Circadian Clockwork to Locomotor Activity in Drosophila.

Neuron 2018 08 26;99(4):768-780.e3. Epub 2018 Jul 26.

Department of Biology, Howard Hughes Medical Institute and National Center for Behavioral Genomics, Brandeis University, 415 South Street, Waltham, MA 02453, USA. Electronic address:

Drosophila NonA and its mammalian ortholog NONO are members of the Drosophila behavior and human splicing (DBHS) family. NONO also has a strong circadian connection: it associates with the circadian repressor protein PERIOD (PER) and contributes to circadian timekeeping. Here, we investigate NonA, which is required for proper levels of evening locomotor activity as well as a normal free-running period in Drosophila. NonA is associated with the positive transcription factor CLOCK/CYCLE (CLK/CYC), interacts directly with complexin (cpx) pre-mRNA, and upregulates gene expression, including the gene cpx. Downregulation of cpx expression in circadian neurons phenocopies NonA downregulation, whereas cpx overexpression rescues the nonA RNAi phenotypes, indicating that cpx is an important NonA target gene. As the cpx protein contributes to proper neurotransmitter and neuropeptide release in response to calcium, these results and others indicate that this control is important for the normal circadian regulation of locomotor activity.
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http://dx.doi.org/10.1016/j.neuron.2018.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442453PMC
August 2018

Citrullination Inactivates Nicotinamide- N-methyltransferase.

ACS Chem Biol 2018 09 8;13(9):2663-2672. Epub 2018 Aug 8.

Department of Biochemistry and Molecular Pharmacology , UMass Medical School , 364 Plantation Street , Worcester , Massachusetts 01605 , United States.

Nicotinamide- N-methyltransferase (NNMT) catalyzes the irreversible methylation of nicotinamide (NAM) to form N-methyl nicotinamide using S-adenosyl methionine as a methyl donor. NNMT is implicated in several chronic disease conditions, including cancers, kidney disease, cardiovascular disease, and Parkinson's disease. Although phosphorylation of NNMT in gastric tumors is reported, the functional effects of this post-translational modification has not been investigated. We previously reported that citrullination of NNMT by Protein Arginine Deiminases abolished its methyltransferase activity. Herein, we investigate the mechanism of inactivation. Using tandem mass spectrometry, we identified three sites of citrullination in NNMT. With this information in hand, we used a combination of site-directed mutagenesis, kinetics, and circular dichoism experiments to demonstrate that citrullination of R132 leads to a structural perturbation that ultimately promotes NNMT inactivation.
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http://dx.doi.org/10.1021/acschembio.8b00578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150842PMC
September 2018

A Quantitative Chemoproteomic Platform to Monitor Selenocysteine Reactivity within a Complex Proteome.

Cell Chem Biol 2018 09 5;25(9):1157-1167.e4. Epub 2018 Jul 5.

Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA. Electronic address:

Mammalian selenocysteine (Sec)-containing proteins, selenoproteins, are important to (patho)physiological processes, including redox homeostasis. Sec residues have been recalcitrant to mass spectrometry-based chemoproteomic methods that enrich for reactive cysteine (Cys) residues with electrophilic chemical probes, despite confirmed reactivity of Sec with these electrophiles. Highly abundant Cys peptides likely suppress low-abundant Sec peptides. By exploiting the decreased pK of Sec relative to Cys, we have developed a chemoproteomic platform that relies on low pH (pH 5.75) electrophile labeling, reducing Cys reactivity and enhancing identification of Sec-containing peptides across mouse tissues and cell lines. The utility of this Sec-profiling platform is underscored by evaluation of the selectivity of auranofin, an inhibitor of the selenoprotein, thioredoxin reductase, against both reactive Cys- and Sec-containing proteins. Platform limitations pertain to the non-physiological low-pH conditions that could perturb protein structure and function. Future work necessitates the discovery of Sec-selective electrophiles that function at physiological pH.
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http://dx.doi.org/10.1016/j.chembiol.2018.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510024PMC
September 2018