RESUMO
Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteínas F-Box/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Desenvolvimento Ósseo , Ilhas de CpG , Proteínas F-Box/química , Proteínas F-Box/genética , Genes Letais , Estudo de Associação Genômica Ampla , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Estrutura Terciária de ProteínaRESUMO
The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.
Assuntos
Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/metabolismo , Succinatos/metabolismo , Alquilação , Animais , Carboxiliases , Bovinos , Cisteína/química , Cisteína/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Retroalimentação Fisiológica , Feminino , Células HEK293 , Humanos , Hidroliases/biossíntese , Interferon beta/imunologia , Interferon beta/farmacologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Proteínas/metabolismo , Ratos , Ratos Wistar , Succinatos/químicaRESUMO
We report a two-step validation approach to evaluate the suitability of metal-binding groups for targeting DNA damage-repair metalloenzymes using model enzyme SNM1A. A fragment-based screening approach was first used to identify metal-binding fragments suitable for targeting the enzyme. Effective fragments were then incorporated into oligonucleotides using the copper-catalysed azide-alkyne cycloaddition reaction. These modified oligonucleotides were recognised by SNM1A at >1000-fold lower concentrations than their fragment counterparts. The exonuclease SNM1A is a key enzyme involved in the repair of interstrand crosslinks, a highly cytotoxic form of DNA damage. However, SNM1A and other enzymes of this class are poorly understood, as there is a lack of tools available to facilitate their study. Our novel approach of incorporating functional fragments into oligonucleotides is broadly applicable to generating modified oligonucleotide structures with high affinity for DNA damage-repair enzymes.
Assuntos
Proteínas de Ciclo Celular , Exodesoxirribonucleases , Exodesoxirribonucleases/metabolismo , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Oligonucleotídeos/químicaRESUMO
Nucleoside analogs show useful bioactive properties. A versatile solid-phase synthesis that readily enables the diversification of thymine-containing nucleoside analogs is presented. The utility of the approach is demonstrated with the preparation of a library of compounds for analysis with SNM1A, a DNA damage repair enzyme that contributes to cytotoxicity. This exploration provided the most promising nucleoside-derived inhibitor of SNM1A to date with an IC50 of 12.3 µM.
Assuntos
Nucleosídeos , Timina , Nucleosídeos/farmacologia , Timina/farmacologia , Técnicas de Síntese em Fase Sólida , Exodesoxirribonucleases/metabolismo , Reparo do DNARESUMO
Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.
Assuntos
Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Animais , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Feminino , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Piperidinas/síntese química , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirazóis/síntese química , Pirimidinas/síntese química , Especificidade por Substrato , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Ciclo Celular , Humanos , Modelos BiológicosRESUMO
Eosin Y was assessed for its ability to induce a thiol-ene dependent protein-protein reaction in a metal-free, oxygen-tolerant, visible light mediated system. Protein-protein coupling efficiency under these mild conditions was comparable to previously reported UV-dependent conditions. The desired thiol-ene reaction was however limited within more complex biological systems.
Assuntos
Cisteína Endopeptidases/química , Enzimas Desubiquitinantes/química , Amarelo de Eosina-(YS)/química , Sondas Moleculares/química , Alcenos/química , Catálise/efeitos da radiação , Cisteína/química , Amarelo de Eosina-(YS)/efeitos da radiação , Células HEK293 , Humanos , Luz , Sondas Moleculares/efeitos da radiaçãoRESUMO
The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase 1 (PRMT1) and symmetric dimethylating PRMT5 and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favors proliferation by antagonizing methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell-cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN downregulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity.
Assuntos
Apoptose , Proliferação de Células , Fator de Transcrição E2F1/metabolismo , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos , Arginina , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Ciclina A/metabolismo , Dano ao DNA , Fator de Transcrição E2F1/genética , Regulação da Expressão Gênica , Humanos , Metilação , Regiões Promotoras Genéticas , Ligação Proteica , Proteína-Arginina N-Metiltransferases/genética , Interferência de RNA , Proteínas Repressoras/genética , Transdução de Sinais , Transcrição Gênica , TransfecçãoRESUMO
SNM1A is a nuclease that is implicated in DNA interstrand crosslink repair and, as such, its inhibition is of interest for overcoming resistance to chemotherapeutic crosslinking agents. However, the number and identity of the metal ion(s) in the active site of SNM1A are still unconfirmed, and only a limited number of inhibitors have been reported to date. Herein, we report the synthesis and evaluation of a family of malonate-based modified nucleosides to investigate the optimal positioning of metal-binding groups in nucleoside-derived inhibitors for SNM1A. These compounds include ester, carboxylate and hydroxamic acid malonate derivatives which were installed in the 5'-position or 3'-position of thymidine or as a linkage between two nucleosides. Evaluation as inhibitors of recombinant SNM1A showed that nine of the twelve compounds tested had an inhibitory effect at 1 mM concentration. The most potent compound contains a hydroxamic acid malonate group at the 5'-position. Overall, our studies advance the understanding of requirements for nucleoside-derived inhibitors for SNM1A and indicate that groups containing a negatively charged group in close proximity to a metal chelator, such as hydroxamic acid malonates, are promising structures in the design of inhibitors.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Exodesoxirribonucleases/antagonistas & inibidores , Nucleosídeos/farmacologia , Compostos Organometálicos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ésteres/química , Ésteres/farmacologia , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Malonatos/química , Malonatos/farmacologia , Estrutura Molecular , Nucleosídeos/química , Compostos Organometálicos/síntese química , Compostos Organometálicos/químicaRESUMO
The Polycomb repressive complexes PRC1 and PRC2 are key mediators of heritable gene silencing in multicellular organisms. Here, we characterise AEBP2, a known PRC2 co-factor which, in vitro, has been shown to stimulate PRC2 activity. We show that AEBP2 localises specifically to PRC2 target loci, including the inactive X chromosome. Proteomic analysis confirms that AEBP2 associates exclusively with PRC2 complexes. However, analysis of embryos homozygous for a targeted mutation of Aebp2 unexpectedly revealed a Trithorax phenotype, normally linked to antagonism of Polycomb function. Consistent with this, we observe elevated levels of PRC2-mediated histone H3K27 methylation at target loci in Aebp2 mutant embryonic stem cells (ESCs). We further demonstrate that mutant ESCs assemble atypical hybrid PRC2 subcomplexes, potentially accounting for enhancement of Polycomb activity, and suggesting that AEBP2 normally plays a role in defining the mutually exclusive composition of PRC2 subcomplexes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteômica/métodos , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Feminino , Histonas/metabolismo , Camundongos , Mutação/genética , Proteínas Nucleares/genética , Complexo Repressor Polycomb 2/genética , Proteínas RepressorasRESUMO
Nine modified nucleosides, incorporating zinc-binding pharmacophores, have been synthesised and evaluated as inhibitors of the DNA repair nuclease SNM1A. The series included oxyamides, hydroxamic acids, hydroxamates, a hydrazide, a squarate ester and a squaramide. A hydroxamic acid-derived nucleoside inhibited the enzyme, offering a novel approach for potential therapeutic development through the use of rationally designed nucleoside derived inhibitors.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Exodesoxirribonucleases/antagonistas & inibidores , Ácidos Hidroxâmicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Exodesoxirribonucleases/metabolismo , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Baculoviruses encode a variety of auxiliary proteins that are not essential for viral replication but provide them with a selective advantage in nature. P10 is a 10-kDa auxiliary protein produced in the very late phase of gene transcription by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The P10 protein forms cytoskeleton-like structures in the host cell that associate with microtubules varying from filamentous forms in the cytoplasm to aggregated perinuclear tubules that form a cage-like structure around the nucleus. These P10 structures may have a role in the release of occlusion bodies (OBs) and thus mediate the horizontal transmission of the virus between insect hosts. Here, using mass spectrometric analysis, it is demonstrated that the C terminus of P10 is phosphorylated during virus infection of cells in culture. Analysis of P10 mutants encoded by recombinant baculoviruses in which putative phosphorylation residues were mutated to alanine showed that serine 93 is a site of phosphorylation. Confocal microscopy examination of the serine 93 mutant structures revealed aberrant formation of the perinuclear tubules. Thus, the phosphorylation of serine 93 may induce the aggregation of filaments to form tubules. Together, these data suggest that the phosphorylation of serine 93 affects the structural conformation of P10.IMPORTANCE The baculovirus P10 protein has been researched intensively since it was first observed in 1969, but its role during viral infection remains unclear. It is conserved in the alphabaculoviruses and expressed at high levels during virus infection. Producing large amounts of a protein is wasteful for the virus unless it is advantageous for the survival of its progeny, and therefore, P10 presents an enigma. As P10 polymerizes to form organized cytoskeletal structures that colocalize with host cell microtubules, the structural relationship of the protein with the host cell may present a key to help understand the function and importance of this protein. This study addresses the importance of the structural changes in P10 during infection and how they may be governed by phosphorylation. The P10 structures affected by phosphorylation are closely associated with the viral progeny and thus may potentially be responsible for its dissemination and survival.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Virais/química , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Análise Mutacional de DNA , Insetos , Espectrometria de Massas , Fosforilação , Conformação Proteica , Multimerização Proteica , Proteínas Virais/genéticaRESUMO
In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role of the proteasome in determining the fate of proteins conjugated to SUMO2 when cells are treated with DNA replication stress conditions. We conducted a quantitative proteomic analysis in a U2OS cell line stably expressing SUMO2(Q87R) tagged with StrepHA in the presence or absence of epoxomicin (EPOX), a proteasome inhibitor. We identified subgroups of putative SUMO2 targets that were either degraded or stabilized by EPOX upon SUMO2 conjugation in response to replication stress. Interestingly, the subgroup of proteins degraded upon SUMO2 conjugation was enriched in proteins playing roles in DNA damage repair and replication, while the proteins stabilized upon SUMOylation were mainly involved in chromatin maintenance. In addition, we identified 43 SUMOylation sites in target proteins, of which 17 are located in the proximity of phosphorylated residues. Considering that DNA replication stress is a major source of genome instability, which is suggested to drive tumorigenesis and possibly aging, our data will facilitate future functional studies in the fields of DNA metabolism and cancer biology.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fenômenos Fisiológicos Celulares/fisiologia , Replicação do DNA/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteômica/métodos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Estresse Fisiológico/fisiologia , Western Blotting , Clonagem Molecular , Citometria de Fluxo , Humanos , Espectrometria de Massas , Microscopia de Fluorescência , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Proteólise/efeitos dos fármacosRESUMO
Affinity-based probes are valuable tools for detecting binding interactions between small molecules and proteins in complex biological environments. Metalloproteins are a class of therapeutically significant biomolecules which bind metal ions as part of key structural or catalytic domains and are compelling targets for study. However, there is currently a limited range of chemical tools suitable for profiling the metalloproteome. Here, we describe the preparation and application of a novel, photoactivatable affinity-based probe for detection of a subset of previously challenging to engage metalloproteins. The probe, bearing an 8-mercaptoquinoline metal chelator, was anticipated to engage several zinc metalloproteins, including the 26S-proteasome subunit Rpn11. Upon translation of the labelling experiment to mammalian cell lysate and live cell experiments, proteomic analysis revealed that several metalloproteins were competitively enriched. The diazirine probe SMK-24 was found to effectively enrich multiple components of the minichromosome maintenance complex, a zinc metalloprotein assembly with helicase activity essential to DNA replication. Cell cycle analysis experiments revealed that HEK293 cells treated with SMK-24 experienced stalling in G0/G1 phase, consistent with inactivation of the DNA helicase complex. This work represents an important contribution to the library of cell-permeable chemical tools for studying a collection of metalloproteins for which no previous probe existed.
RESUMO
Quick and clean: A method for Pd-catalyzed Suzuki-Miyaura cross-coupling to iododeoxyuridine (IdU) in DNA is described. Key to the reactivity is the choice of the ligand and the buffer. A covalent [Pd]-DNA intermediate was isolated and characterized. Photocrosslinking probes were generated to trap proteins that bind to epigenetic DNA modifications.
Assuntos
DNA/química , Sondas Moleculares/química , Oligonucleotídeos/química , Catálise , DNA/genética , Idoxuridina/química , Oligonucleotídeos/genética , Paládio/química , Marcadores de Fotoafinidade/químicaRESUMO
Novel ubiquitin-based active site probes including a fluorescent tag have been developed and evaluated. A new, functionalizable electrophilic trap is utilized allowing for late stage diversification of the probe. Attachment of fluorescent dyes allowed direct detection of endogenous deubiquitinating enzyme (DUB) activities in cell extracts by in-gel fluorescence imaging.
Assuntos
Domínio Catalítico , Endopeptidases/química , Endopeptidases/metabolismo , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/metabolismo , Química Click , Corantes Fluorescentes/química , Células HEK293 , Humanos , Modelos Moleculares , Ubiquitina/química , Ubiquitina/metabolismoRESUMO
Certain cancers exhibit upregulation of DNA interstrand crosslink repair pathways, which contributes to resistance to crosslinking chemotherapy drugs and poor prognoses. Inhibition of enzymes implicated in interstrand crosslink repair is therefore a promising strategy for improving the efficacy of cancer treatment. One such target enzyme is SNM1A, a zinc co-ordinating 5'-3' exonuclease. Previous studies have demonstrated the feasibility of inhibiting SNM1A using modified nucleosides appended with zinc-binding groups. In this work, we sought to develop more effective SNM1A inhibitors by exploiting interactions with the phosphate-binding pocket adjacent to the enzyme's active site, in addition to the catalytic zinc ions. A series of nucleoside derivatives bearing phosphate moieties at the 5'-position, as well as zinc-binding groups at the 3'-position, were prepared and tested in gel-electrophoresis and real-time fluorescence assays. As well as investigating novel zinc-binding groups, we found that incorporation of a 5'-phosphate dramatically increased the potency of the inhibitors.
Assuntos
Exodesoxirribonucleases , Nucleosídeos , Reparo do DNA , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Nucleosídeos/farmacologia , Fosfatos , Fosforilação , Zinco/farmacologiaRESUMO
Over the past two decades, activity-based probes have enabled a range of discoveries, including the characterization of new enzymes and drug targets. However, their suitability in some labeling experiments can be limited by nonspecific reactivity, poor membrane permeability, or high toxicity. One method for overcoming these issues is through the development of "inducible" activity-based probes. These probes are added to samples in an unreactive state and require in situ transformation to their active form before labeling can occur. In this Review, we discuss a variety of approaches to inducible activity-based probe design, different means of probe activation, and the advancements that have resulted from these applications. Additionally, we highlight recent developments which may provide opportunities for future inducible activity-based probe innovations.
Assuntos
Sondas Moleculares/química , Proteínas/química , Corantes Fluorescentes/química , Processos Fotoquímicos , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Deubiquitinating enzymes (DUBs) are known to have numerous important interactions with the ubiquitin cascade and their dysregulation is associated with several diseases, including cancer and neurodegeneration. They are an important class of enzyme, and activity-based probes have been developed as an effective strategy to study them. Existing activity-based probes that target the active site of these enzymes work via nucleophilic mechanisms. We present the development of latent ubiquitin-based probes that target DUBs via a site selective, photoinitiated radical mechanism. This approach differs from existing photocrosslinking probes as it requires a free active site cysteine. In contrast to existing cysteine reactive probes, control over the timing of the enzyme-probe reaction is possible as the alkene warhead is completely inert under ambient conditions, even upon probe binding. The probe's reactivity has been demonstrated against recombinant DUBs and to capture endogenous DUB activity in cell lysate. This allows more finely resolved investigations of DUBs.
RESUMO
A systematically varied series of conformationally restricted ketones, readily prepared from N-acetyl-D-glucosamine, were tested against representative olefins as asymmetric epoxidation catalysts showing useful selectivities against terminal olefins and, in particular, typically difficult 2,2-disubstituted terminal olefins.