Protein arginine methyltransferase 1 (PRMT1) catalyzes the mono- and dimethylation of certain protein arginine residues. Although this posttranslational modification has been implicated in many physiological processes, the molecular basis for PRMT1 substrate recognition is poorly understood. Most modified arginine residues in known PRMT1 substrates reside in repeating "RGG" sequences. However, PRMT1 also specifically methylates Arg3 of histone H4 in a region that is not glycine-arginine rich, suggesting that PRMT1 substrates are not limited to proteins bearing "RGG" sequences. Because a systematic evaluation of PRMT1 substrate specificity has not been performed, it is unclear if the "RGG" sequence accurately represents the consensus target for PRMT1. Using a focused peptide library based on a sequence derived from the in vivo substrate fibrillarin we observed that PRMT1 methylated substrates that had amino acid residues other than glycine in the "RX (1)" and "RX (1)X (2)" positions. Importantly, eleven additional PRMT1 substrate sequences were identified. Our results also illustrate that the two residues on the N-terminal side of the modification site are important and need not both be glycine. PRMT1 methylated the eukaryotic initiation factor 4A1 (eIF4A1) protein, which has a single "RGG" sequence. Methylation of eIF4A1 and the similar eIF4A3 could be affected using single site mutations adjacent to the modification site, demonstrating the importance of amino acid sequence in PRMT1 protein substrates. Dimethylation of the parent library peptide was shown to occur through a dissociative mechanism. In summary, PRMT1 selectively recognizes a set of amino acid sequences in substrates that extend beyond the "RGG" paradigm.
Protein arginine methyltransferases (PRMTs) aid in the regulation of many biological processes. Accurate control of PRMT activity includes recognition of specific arginyl groups within targeted proteins and the generation of the correct level of methylation, none of which are fully understood. The predominant PRMT in vivo, PRMT1, has wide substrate specificity and is capable of both mono- and dimethylation, which can induce distinct biological outputs. What regulates the specific methylation pattern of PRMT1 in vivo is unclear. We report that PRMT1 methylates a multisite peptide substrate in a nonstochastic manner, with less C-terminal preference, consistent with the methylation patterns observed in vivo. With a single targeted arginine, PRMT1 catalyzed the dimethylation in a semiprocessive manner. The degree of processivity is regulated by substrate sequences. Our results identify a novel substrate-induced mechanism for modulating PRMT1 product specificity. Considering the numerous physiological PRMT1 substrates, as well as the distinct biological outputs of mono- and dimethylation products, such fine-tuned regulation would significantly contribute to the accurate product specificity of PRMT1 in vivo and the proper transmission of biochemical information.
Isoaspartate formation is a ubiquitous post-translation modification arising from spontaneous asparagine deamidation or aspartate isomerization. The formation of isoaspartate inserts a methylene group into the protein backbone, generating a "kink", and may drastically alter protein structure and function, thereby playing critical roles in a myriad of biological processes, human diseases, and protein pharmaceutical development. Herein, we report a chemo-enzymatic detection method for the isoaspartate protein, which in particular allows the affinity enrichment of isoaspartate-containing proteins. In the initial step, protein isoaspartate methyltransferase selectively converts isoaspartates into the corresponding methyl esters. Subsequently, the labile methyl ester is trapped by strong nucleophiles in aqueous solutions, such as hydrazines to form hydrazides. The stable hydrazide products can be analyzed by standard proteomic techniques, such as matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. Furthermore, the chemical trapping step allows us to introduce several tagging strategies for product identification and quantification, such as UV-vis and fluorescence detection through a dansyl derivative. Most significantly, the hydrazide product can be enriched by affinity chromatography using aldehyde resins, thus drastically reducing sample complexity. Our method hence represents the first technique for the affinity enrichment of isoaspartyl proteins and should be amendable to the systematic and comprehensive characterization of isoaspartate, particularly in complex systems.
Elevated blood levels of homocysteine (Hcy), hyperhomocysteinemia or homocystinuria, have been associated with various diseases and conditions. Homocysteine thiolactone (Hcy TL) is a metabolite of Hcy and reacts with amine groups in proteins to form stable amides, homocystamides or N-homocysteinylated proteins. It has been proposed that protein N-homocysteinylation contributes to the cytotoxicity of elevated Hcy. Due to its heterogeneity and relatively low abundance, detection of this post-translational modification remains challenging. On the other hand, the gamma-aminothiol group in homocystamides imparts different chemical reactivities than the native proteins. Under mildly acidic conditions, gamma-aminothiols irreversibly and stoichiometrically react with aldehydes to form stable 1,3-thiazines, whereas the reversible Schiff base formation between aldehydes and amino groups in native proteins is markedly disfavored due to protonation of amines. As such, we have developed highly selective chemical methods to derivatize N-homocysteinylated proteins with various aldehyde tags, thereby facilitating the subsequent analyses. For instance, fluorescent or biotin tagging coupled with gel electrophoresis permits quantification and global profiling of complex biological samples, such as hemoglobin and plasma from rat, mouse and human; affinity enrichment with aldehyde resins drastically reduces sample complexity. In addition, different reactivities of lysine residues in hemoglobin towards Hcy TL were observed.
S-Adenosylmethionine (AdoMet or SAM)-dependent methyltransferases belong to a large and diverse family of group-transfer enzymes that perform vital biological functions on a host of substrates. Despite the progress in genomics, structural proteomics and computational biology, functional annotation of methyltransferases remains a challenge. Herein, we report the synthesis and activity of a new AdoMet analog functionalized with a ketone group. Using catechol O-methyltransferase (COMT, EC 2.1.1.6) and thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) as model enzymes, this robust and readily accessible analog displays kinetic parameters that are comparable to AdoMet and exhibits multiple turnovers with enzyme. More importantly, this AdoMet surrogate displays the same substrate specificity as the natural methyl donor. Incorporation of the ketone group allows for subsequent modification via bio-orthogonal labeling strategies and sensitive detection of the tagged ketone products. Hence, this AdoMet analog expands the toolbox available to interrogate the biochemical functions of methyltransferases.
Halogenated furanones, a group of natural products initially isolated from marine red algae, are known to inhibit bacterial biofilm formation, swarming, and quorum sensing. However, their molecular targets and the precise mode of action remain elusive. Herein, we show that a naturally occurring brominated furanone covalently modifies and inactivates LuxS (S-ribosylhomocysteine lyase, EC 4.4.1.21), the enzyme which produces autoinducer-2 (AI-2).
Aldo-keto reductase 1C3 (AKR1C3), also known as type 5 17 β-hydroxysteroid dehydrogenase, is responsible for intratumoral androgen biosynthesis, contributing to the development of castration-resistant prostate cancer (CRPC) and eventual chemotherapeutic failure. Significant upregulation of AKR1C3 is observed in CRPC patient samples and derived CRPC cell lines. As AKR1C3 is a downstream steroidogenic enzyme synthesizing intratumoral testosterone (T) and 5α-dihydrotestosterone (DHT), the enzyme represents a promising therapeutic target to manage CRPC and combat the emergence of resistance to clinically employed androgen deprivation therapy. Herein, we demonstrate the antineoplastic activity of a potent, isoform-selective and hydrolytically stable AKR1C3 inhibitor ()-3-(4-(3-methylbut-2-en-1-yl)-3-(3-phenylpropanamido)phenyl)acrylic acid (), which reduces prostate cancer cell growth and and sensitizes CRPC cell lines (22Rv1 and LNCaP1C3) toward the antitumor effects of enzalutamide. Crucially, does not induce toxicity in nonmalignant WPMY-1 prostate cells nor does it induce weight loss in mouse xenografts. Moreover, reduces androgen receptor (AR) transactivation and prostate-specific antigen expression levels in CRPC cell lines indicative of a therapeutic effect in prostate cancer. Combination studies of with enzalutamide reveal a very high degree of synergistic drug interaction that induces significant reduction in prostate cancer cell viability via apoptosis, resulting in>200-fold potentiation of enzalutamide action in drug-resistant 22Rv1 cells. These results demonstrate a promising therapeutic strategy for the treatment of drug-resistant CRPC that invariably develops in prostate cancer patients following initial treatment with AR antagonists such as enzalutamide. .
Aldo–keto reductase 1C3 (AKR1C3) catalyzes the synthesis of 9α,11β-prostaglandin (PG) F2α and PGF2α prostanoids that sustain the growth of myeloid precursors in the bone marrow. The enzyme is overexpressed in acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (T-ALL). Moreover, AKR1C3 confers chemother-apeutic resistance to the anthracyclines: first-line agents for the treatment of leukemias. The highly homologous isoforms AKR1C1 and AKR1C2 inactivate 5α-dihydrotestosterone, and their inhibition would be undesirable. We report herein the identification of AKR1C3 inhibitors that demonstrate exquisite isoform selectivity for AKR1C3 over the other closely related isoforms to the order of >2800-fold. Biological evaluation of our isoform-selective inhibitors revealed a high degree of synergistic drug action in combination with the clinical leukemia therapeutics daunorubicin and cytarabine in in vitro cellular models of AML and primary patient-derived T-ALL cells. Our developed compounds exhibited >100-fold dose reduction index that results in complete resensitization of a daunorubicin-resistant AML cell line to the chemotherapeutic and >100-fold dose reduction of cytarabine in both AML cell lines and primary T-ALL cells.
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