Covalent hitchhikers guide proteins to the nucleus.

In this issue of Cell Chemical Biology, Peng and Weerapana report the combination of chemoproteomic and proximity-based labeling approaches to identify cysteines in nuclear proteins that are reactive toward electrophilic probe compounds. They apply this novel technology to identify proteins that are localized to the nucleus and chromatin upon probe labeling.

The semisynthesis of site-specifically modified histones and histone-based probes of chromatin-modifying enzymes.

Histone post-translational modifications (PTMs) on lysine residues, including methylation, ubiquitylation, and sumoylation, have been studied using semisynthetic histones reconstituted into nucleosomes. These studies have revealed the in vitro effects of histone PTMs on chromatin structure, gene transcription, and biochemical crosstalk. However, the dynamic and transient nature of most enzyme-chromatin interactions poses a challenge toward identifying specific enzyme-substrate interactions.

Total chemical synthesis of sumoylated histone H4 reveals negative biochemical crosstalk with histone ubiquitylation.

An efficient total chemical synthesis of site-specifically sumoylated histone H4 was undertaken to generate homogenously modified mononucleosomes. These were tested as substrates in biochemical assays with the histone H2B-specific ubiquitin ligases Rad6 and Bre1, which revealed the strong inhibition of H2B ubiquitylation by SUMO. This novel negative biochemical crosstalk between SUMO and ubiquitin was also confirmed to exist in human cells.

Peptide and protein chemistry approaches to study the tumor suppressor protein p53.

The tumor suppressor and master gene regulator protein p53 has been the subject of intense investigation for several decades due to its mutation in about half of all human cancers. However, mechanistic studies of p53 in cells are complicated by its many dynamic binding partners and heterogeneous post-translational modifications. The design of therapeutics that rescue p53 functions in cells requires a mechanistic understanding of its protein-protein interactions in specific protein complexes and identifying changes in p53 activity by diverse post-translational modifications.

Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts.

The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear.

Development of Tyrphostin Analogues to Study Inhibition of the Mycobacterium tuberculosis Pup Proteasome System*.

Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera.

BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1.

Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes.

Tackling a Curious Case: Generation of Charge-Tagged Guanosine Radicals by Gas-Phase Electron Transfer and Their Characterization by UV-vis Photodissociation Action Spectroscopy and Theory.

We report the generation of gas-phase riboguanosine radicals that were tagged at ribose with a fixed-charge 6-(trimethylammonium)hexane-1-aminocarbonyl group. The radical generation relied on electron transfer from fluoranthene anion to noncovalent dibenzocrown-ether dication complexes which formed nucleoside cation radicals upon one-electron reduction and crown-ether ligand loss. The cation radicals were characterized by collision-induced dissociation (CID), photodissociation (UVPD), and UV-vis action spectroscopy.

Trimethylation of the R5 Silica-Precipitating Peptide Increases Silica Particle Size by Redirecting Orthosilicate Binding.

The unmodified R5 peptide from silaffin in the diatom Cylindrotheca fusiformis rapidly precipitates silica particles from neutral aqueous solutions of orthosilicic acid. A range of post-translational modifications found in R5 contribute toward tailoring silica morphologies in a species-specific manner. We investigated the specific effect of R5 lysine side-chain trimethylation, which adds permanent positive charges, on silica particle formation.

Nucleosome Binding by the Lysine Specific Demethylase 1 (LSD1) Enzyme Enables Histone H3 Demethylation.

The essential human enzyme lysine specific demethylase 1 (LSD1) silences genes by demethylating mono- and dimethylated lysine 4 in histone H3 (H3K4me1/2). Studies of the minimal requirements for LSD1 activity are complicated by the heterogeneity of histone modification states in cells. We overcame this challenge by generating homogeneous mononucleosome substrates containing semisynthetic H3K4me2.

Facile Semisynthesis of Ubiquitylated Peptides with the Ligation Auxiliary 2-Aminooxyethanethiol.

The posttranslational modification of cellular proteins by ubiquitin (Ub), called ubiquitylation, is indispensable for the normal growth and development of eukaryotic organisms. In order to conduct studies that elucidate the precise mechanistic roles for Ub, access to site-specifically and homogenously ubiquitylated proteins and peptides is critical. However, the low abundance, heterogeneity, and dynamic nature of protein ubiquitylation are significant limitations toward such studies.

Structural Basis of H2B Ubiquitination-Dependent H3K4 Methylation by COMPASS.

The COMPASS (complex of proteins associated with Set1) complex represents the prototype of the SET1/MLL family of methyltransferases that controls gene transcription by H3K4 methylation (H3K4me). Although H2B monoubiquitination (H2Bub) is well known as a prerequisite histone mark for COMPASS activity, how H2Bub activates COMPASS remains unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of an extended COMPASS catalytic module (CM) bound to the H2Bub and free nucleosome.

A clickable glutamine (CliQ) derivative for the traceless reversible modification of peptides and proteins.

The Cu(i)-mediated click reaction of proteins with affinity tags enables their selective isolation from complex mixtures. However, irreversible protein modification limits the interpretation of results from subsequent biophysical and biochemical assays. We report a facile and modular chemical strategy to reversibly modify peptides and proteins with biotin and FLAG affinity tags at a clickable glutamine (CliQ) residue.

Crystal Structure of the COMPASS H3K4 Methyltransferase Catalytic Module.

The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1.

Studies of biochemical crosstalk in chromatin with semisynthetic histones.

Reversible post-translational modifications of histone proteins in eukaryotic chromatin are closely tied to gene function and cellular development. Specific combinations of histone modifications, or marks, are implicated in distinct DNA-templated processes mediated by a range of chromatin-associated enzymes that install, erase and interpret the histone code. Mechanistic studies of the precise biochemical relationship between sets of marks and their effects on chromatin function are significantly complicated by the dynamic nature and heterogeneity of marks in cellular chromatin.

BARD1 is necessary for ubiquitylation of nucleosomal histone H2A and for transcriptional regulation of estrogen metabolism genes.

Missense mutations that disrupt the RING domain of the tumor suppressor gene lead to increased risk of breast and ovarian cancer. The BRCA1 RING domain is a ubiquitin ligase, whose structure and function rely critically on forming a heterodimer with BARD1, which also harbors a RING domain. The function of the BARD1 RING domain is unknown.

Biological consequences of improving the structural stability of hairpins that have antimicrobial activity.

Designing new antimicrobial peptides (AMPs) focuses heavily on the activity of the peptide and less on the elements that stabilize the secondary structure of these peptides. Studies have shown that improving the structure of naturally occurring AMPs can affect activity and so here we explore the relationship between structure and activity of two non-naturally occurring AMPs. We have used a backbone-cyclized peptide as a template and designed an uncyclized analogue of this peptide that has antimicrobial activity.

Chemically Sumoylated Histone H4 Stimulates Intranucleosomal Demethylation by the LSD1-CoREST Complex.

Lysine-specific demethylase 1 (LSD1) downregulates eukaryotic gene activity by demethylating mono- and dimethylated Lys4 in histone H3. Elucidating the biochemical crosstalk of LSD1 with histone post-translational modifications (PTMs) is essential for developing LSD1-targeted therapeutics in human cancers. We interrogated the small ubiquitin-like modifier (SUMO)-driven regulation of LSD1 activity with semisynthetic nucleosomes containing site-specifically methylated and sumoylated histones.

A Facile N-Mercaptoethoxyglycinamide (MEGA) Linker Approach to Peptide Thioesterification and Cyclization.

The C-terminal electrophilic activation of peptides by α-thioesterification requires strongly acidic conditions or complex chemical manipulations, which ultimately limit functional group compatibility and broad utility. Herein, we report a readily accessible N-mercaptoethoxyglycinamide (MEGA) solid-phase linker for the facile synthesis of latent peptide α-thioesters. Incubating peptide-MEGA sequences with 2-mercaptoethanesulfonic acid at mildly acidic pH yielded α-thioesters that were directly used in NCL without purification.

Hairpin structure stability plays a role in the activity of two antimicrobial peptides.

Many naturally occurring antimicrobial peptides (AMPs) are amphipathic with a β-hairpin conformation stabilized by cross-strand disulfides across the associated β-strands. Here, we show that the disulfides are not essential. Other structuring means such as better β-turns and noncovalent cross-strand interactions can, with proper design, replace the disulfides with no loss in antimicrobial activity.

Selenocysteine as a Latent Bioorthogonal Electrophilic Probe for Deubiquitylating Enzymes.

Deubiquitylating enzymes (DUBs) remove ubiquitin (Ub) from various cellular proteins and render eukaryotic ubiquitylation a dynamic process. The misregulation of protein ubiquitylation is associated with many human diseases, and there is an urgent need to identify specific DUBs associated with therapeutically relevant targets of Ub. We report the development of two facile selenocysteine-based strategies to generate the DUB probe dehydroalanine (Dha).

Aromatic thiol-mediated cleavage of N-O bonds enables chemical ubiquitylation of folded proteins.

Access to protein substrates homogenously modified by ubiquitin (Ub) is critical for biophysical and biochemical investigations aimed at deconvoluting the myriad biological roles for Ub. Current chemical strategies for protein ubiquitylation, however, employ temporary ligation auxiliaries that are removed under harsh denaturing conditions and have limited applicability. We report an unprecedented aromatic thiol-mediated N-O bond cleavage and its application towards native chemical ubiquitylation with the ligation auxiliary 2-aminooxyethanethiol.

Allosteric Activation of Ubiquitin-Specific Proteases by β-Propeller Proteins UAF1 and WDR20.

Ubiquitin-specific proteases (USPs) constitute the largest family of deubiquitinating enzymes, whose catalytic competency is often modulated by their binding partners through unknown mechanisms. Here we report on a series of crystallographic and biochemical analyses of an evolutionarily conserved deubiquitinase, USP12, which is activated by two β-propeller proteins, UAF1 and WDR20. Our structures reveal that UAF1 and WDR20 interact with USP12 at two distinct sites far from its catalytic center.

All about that Amide Bond: The Sixth Chemical Protein Synthesis (CPS) Meeting.

Endless potential: The sixth Chemical Protein Synthesis Meeting, held recently in St. Augustine, Florida, showed the potential of peptide and protein chemistry when applied toward understanding and controlling complex biological processes. This report highlights the diverse and cutting-edge protein chemistry presented at the meeting.

A time-resolved Förster resonance energy transfer assay to measure activity of the deamidase of the prokaryotic ubiquitin-like protein.

The modification of proteins in Mycobacterium tuberculosis (Mtb) by the prokaryotic ubiquitin-like protein (Pup) targets them for degradation by mycobacterial proteasomes. Although functionally similar to eukaryotic deubiquitylating enzymes, the deamidase of Pup, called Dop, has no known mammalian homologs. Because Dop is necessary for persistent infection by Mtb, its selective inhibition holds potential for tuberculosis therapy.