A short ORF-encoded transcriptional regulator.

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MSn. The MSn-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.

Metal-free aminofluorination of α-diazo 2H-benzopyran-4-one: convenient access to ß-fluoramides.

We have developed a convenient synthesis of a series of ß-fluoramides in 65% yield. The process involved a tandem fluorination/Ritter reaction to synthesize ß-fluoramides using α-diazo 2H-benzopyran-4-one compounds. Selectfluor was used as the electrophilic fluoride source in acetonitrile to build the ß-fluorinated quaternary carbon center and amide derivatives of 2H-benzopyran-4-one in one step. The products N-(2-(2-fluoro-2,3-dihydro-3-oxobenzofuran-2-yl)propan-2-yl)acetamides were a series of bifunctional compounds with a 2-fluoro-2,3-dihydro-3-oxobenzofuran motif and amide groups.

Photo-crosslinking: An Emerging Chemical Tool for Investigating Molecular Networks in Live Cells.

Studying protein-protein interactions (PPIs) is useful for understanding cellular functions and mechanisms. Evaluating these PPIs under conditions as similar as possible to native conditions can be achieved using photo-crosslinking methods because of their on-demand ability to generate reactive species in situ by irradiation with UV light. Various fusion tag, metabolic incorporation, and amber codon suppression approaches using various crosslinkers containing aryl azide, benzophenone, and diazirines have been applied in live cells. Mass spectrometry and immunological techniques are used to identify crosslinked proteins based on their capture transient and context-dependent interactions. Herein we discuss various incorporation methods and crosslinkers that have been used for interactome mapping in live cells.

Method for Quantification of Ribonucleotides and Deoxyribonucleotides in Human Cells Using (Trimethylsilyl)diazomethane Derivatization Followed by Liquid Chromatography-Tandem Mass Spectrometry.

Investigation into intracellular ribonucleotides (RNs) and deoxyribonucleotides (dRNs) is important for studies of the mechanism of many biological processes, such as RNA and DNA synthesis and DNA repair, as well as metabolic and therapeutic efficacy of nucleoside analogues. However, current methods are still unsatisfactory for determination of nucleotides in complex matrixes. Here we describe a novel method for the determination of RN and dRN pools in cells based on fast derivatization with (trimethylsilyl)diazomethane (TMSD) followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Derivatization was accomplished in 3 min, and each derivatized nucleotide not only had a sufficient retention on reversed-phase column by introduction of methyl groups but also exhibited a unique ion transition which consequently eliminated mutual interference in LC-MS/MS. Chromatographic separation was performed on a C18 column with a simple acetonitrile-water gradient elution system, which avoided contamination and ion suppression caused by ion-pairing reagents. The developed method was fully validated and applied to the analysis of RNs and dRNs in cell samples. Moreover, results demonstrated that the applicability of this method could be extended to nucleoside analogues and their metabolites and could facilitate many applications in future studies.

Subunit interactions as mediated by "non-interface" residues in living cells for multiple homo-oligomeric proteins.

Protein-protein interaction, including protein homo-oligomerization, is commonly believed to occur through a specific interface made of a limited number of amino acid residues. Here our systematic in vivo photo-crosslinking analysis via genetically incorporated unnatural amino acids unexpectedly shows that the dimerization of HdeA, an acid stress chaperone, is mediated by the residues along its whole polypeptide. These include those "forbidden" residues that are far away from the dimerization interface as judged according to the reported 3-D structure. We demonstrate that such dimerization, though intriguing, is neither a result of protein over-expression nor of any structural disturbance caused by the residue replacement. Similar unexpected dimerization also occurs for two other oligomeric proteins, IbpB (a molecular chaperone existing as polydispersed oligomers in vitro) and DegP (a protease existing as hexamers in vitro). In contrast to these three proteins, dimerization of a few other oligomeric proteins (e.g., OmpF, LamB, SurA, FtsZ and FkpA) that we similarly examined in living cells seems to be mediated only by specific residues. Together, our unexpected observations suggest that, for some oligomeric proteins such as HdeA, IbpB and DegP, their subunit interactions in living cells can also be mediated by residues other than those located at the interfaces as revealed by in vitro structure determination. Our observations might be partially explained by the formation of "encounter complex" or by protein conformational dynamics. Our findings provide new insights on understanding protein-protein interactions and encounter complex formation in living cells.

Photolytic Labeling To Quantify Peptide-Water Interactions in Lyophilized Solids.

Interactions of a lyophilized peptide with water and excipients in a solid matrix were explored using photolytic labeling. A model peptide "KLQ" (Ac-QELHKLQ-NHCH3) was covalently labeled with NHS-diazirine (succinimidyl 4,4'-azipentanoate), and the labeled peptide (KLQ-SDA) was formulated and exposed to UV light in both solution and lyophilized solids. Solid samples contained the following excipients at a 1:400 molar ratio: sucrose, trehalose, mannitol, histidine, or arginine. Prior to UV exposure, the lyophilized solids were exposed to various relative humidity (RH) environments (8, 13, 33, 45, and 78%), and the resulting solid moisture content (Karl Fischer titration) and glass transition temperature ( Tg; differential scanning calorimetry, DSC) were measured. To initiate photolytic labeling, solution and solid samples were exposed to UV light at 365 nm for 30 min. Photolytic-labeling products were quantified using reversed-phase high-performance liquid chromatography (rp-HPLC) and mass spectrometry (MS). In lyophilized solids, studies excluding oxygen and using H218O confirmed that the source of oxygen in KLQ adducts with a mass increase of 18 amu are attributable to reaction with water, while those with a mass increase of 16 amu are not attributable to reaction with either water or molecular oxygen. In solids containing sucrose or trehalose, peptide-excipient adducts decreased with increasing solid moisture content, while peptide-water adducts increased only at lower RH exposure and then plateaued, in partial agreement with the water replacement hypothesis.

Profiling the Nucleobase and Structure Selectivity of Anticancer Drugs and other DNA Alkylating Agents by RNA Sequencing.

Drugs that covalently modify DNA are components of most chemotherapy regimens, often serving as first-line treatments. Classically, the reactivity and selectivity of DNA alkylating agents has been determined in vitro with short oligonucleotides. A statistically sound analysis of sequence preferences of alkylating agents is untenable with serial analysis methods because of the combinatorial explosion of sequence possibilities. Next-generation sequencing (NGS) is ideally suited for the broad characterization of sequence or structure selectivities because it analyzes many sequences at once. Herein, NGS is used to report on the chemoselectivity of alkylating agents on RNA and this technology is applied to the previously uncharacterized alkylating agent trimethylsilyl diazomethane.

Conditional Chaperone-Client Interactions Revealed by Genetically Encoded Photo-cross-linkers.

The cell envelope is an integral and essential component of Gram-negative bacteria. As the front line during host-pathogen interactions, it is directly challenged by host immune responses as well as other harsh extracellular stimuli. The high permeability of the outer-membrane and the lack of ATP energy system render it difficult to maintain important biological activities within the periplasmic space under stress conditions. The HdeA/B chaperone machinery is the only known acid resistant system found in bacterial periplasm, enabling enteric pathogens to survive through the highly acidic human stomach and establish infections in the intestine. These two homologous chaperones belong to a fast growing family of conditionally disordered chaperones that conditionally lose their well-defined three-dimensional structures to exert biological activities. Upon losing ordered structures, these proteins commit promiscuous binding of diverse clients in response to environmental stimulation. For example, HdeA and HdeB are well-folded inactive dimers at neutral pH but become partially unfolded to protect a wide array of acid-denatured proteins upon acid stress. Whether these conditionally disordered chaperones possess client specificities remains unclear. This is in part due to the lack of efficient tools to investigate such versatile and heterogeneous protein-protein interactions under living conditions. Genetically encoded protein photo-cross-linkers have offered a powerful strategy to capture protein-protein interactions, showing great potential in profiling protein interaction networks, mapping binding interfaces, and probing dynamic changes in both physiological and pathological settings. Despite great success, photo-cross-linkers that can simultaneously capture the promiscuous binding partners and directly identify the interaction interfaces remain technically challenging. Furthermore, methods for side-by-side profiling and comparing the condition-dependent client pools from two homologous chaperones are lacking. Herein, we introduce our recent efforts in developing a panel of versatile genetically encoded photo-cross-linkers to study the disorder-mediated chaperone-client interactions in living cells. In particular, we have developed a series of proteomic-based strategies relying on these new photo-cross-linkers to systematically compare the client profiles of HdeA and HdeB, as well as to map their interaction interfaces. These studies revealed the mode-of-action, particularly the client specificity, of these two conditionally disordered chaperones. In the end, some recent elegant work from other groups that applied the genetically encoded photo-cross-linking strategy to illuminate important protein-protein interactions within bacterial cell envelope is also discussed.

Photo-lysine captures proteins that bind lysine post-translational modifications.

Post-translational modifications (PTMs) have key roles in regulating protein-protein interactions in living cells. However, it remains a challenge to identify these PTM-mediated interactions. Here we develop a new lysine-based photo-reactive amino acid, termed photo-lysine. We demonstrate that photo-lysine, which is readily incorporated into proteins by native mammalian translation machinery, can be used to capture and identify proteins that recognize lysine PTMs, including 'readers' and 'erasers' of histone modifications.

Relative Quantification of Phospholipids Based on Isotope-Labeled Methylation by Nanoflow Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry: Enhancement in Cardiolipin Profiling.

In this study, lipid analysis based on isotope-labeled methlylation (ILM) was performed by nanoflow ultrahigh performance liquid chromatography-eletrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) for enhanced detection and quantification of targeted phospholipids. ILM depends on methylation of phosphate groups by (trimethylsilyl)diazomethane, and the ILM based quantitation with reversed phase nUPLC-ESI-MS/MS provides advantages in PL profiling such as enhanced detectability of methylated PLs owing to increased hydrophobicity and substantial increase in resolution due to the increase of retention. Efficacy of ILM in nUPLC-ESI-MS/MS analysis was evaluated in the selected reaction monitoring (SRM) method by varying the mixing ratio of H-/D-methylated PL standards, which resulted in the successful quantification of 24 species, including phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), ceramide-1-phosphate (Cer1P), phosphoinositides, and cardiolipin (CL), with ∼6.6% variation in the calculated ratio of H-/D-methylated PLs. The method was applied to the lipid extracts from a DU145 cell line after D-allose treatment, resulting in the quantification of 83 PLs of which results were not statistically different from those obtained by conventional quantification methods. Morever, detection and quantification of CLs and PAs were evidenced to be highly effective when used with the ILM method as 43 CLs and 20 PAs from cellular lipid extracts were analyzed while only 18 CLs and 12 PAs were identified when conventional methods were carried out. This proves the ILM combined with LC-MS to be a promising method for analysis of the aforementioned classes of lipids. Overall, the study highlighted the applicability of targeted quantification by the ILM method in lipidomic analysis and demonstrated an improvement in the detection of less abundant anionic PLs.

Re-exploration of the Codon Context Effect on Amber Codon-Guided Incorporation of Noncanonical Amino Acids in Escherichia coli by the Blue-White Screening Assay.

The effect of codon context on amber codon-guided incorporation of noncanonical amino acids (NAAs) has been previously examined by antibiotic selection. Here, we re-explored this effect by screening a library in which three nucleotides upstream and downstream of the amber codon were randomised, and inserted within the lacZ-α gene. Thousands of clones were obtained and distinguished by the depth of blue colour upon exposure to X-gal. Large-scale sequencing revealed remarkable preferences in nucleotides downstream of the amber codon, and moderate preferences for upstream nucleotides. Nucleotide preference was quantified by a dual-luciferase assay, which verified that the optimum context for NAA incorporation, AATTAGACT, was applicable to different proteins. Our work provides a general guide for engineering amber codons into genes of interest in bacteria.

Synthesis of Arylazide- and Diazirine-Containing CrAsH-EDT2 Photoaffinity Probes.

Two photo-crosslinking biarsenical (CrAsH-EDT2 )-modified probes were synthesized that are expected to be useful tools for tetracysteine-labeled proteins to facilitate the co-affinity purification of their DNA binding sequences and interacting proteins. In addition, improvements for the synthesis of CrAsH-EDT2 and N(1) -(4-azido-2-nitrophenyl)hexane-1,6-diamine are reported. Both photoprobes effectively entered HeLa cells (and the nucleus) and were dependent on the tetracysteine motif in recombinant DMRT1 (doublesex and Mab3-related transcription factor) to induce fluorescence, suggesting that their crosslinking abilities can be exploited for the identification of nucleic acids and proteins associated with a protein of interest.

Active site labeling of cysteine cathepsins by a straightforward diazomethylketone probe derived from the N-terminus of human cystatin C.

We designed a straightforward biotinylated probe using the N-terminal substrate-like region of the inhibitory site of human cystatin C as a scaffold, linked to the thiol-specific reagent diazomethylketone group as a covalent warhead (i.e. Biot-(PEG)2-Ahx-LeuValGly-DMK). The irreversible activity-based probe bound readily to cysteine cathepsins B, L, S and K. Moreover affinity labeling is sensitive since active cathepsins were detected in the nM range using an ExtrAvidin-peroxidase conjugate for disclosure. Biot-(PEG)2-Ahx-LeuValGly-DMK allowed a slightly more pronounced labeling for cathepsin S with a compelling second-order rate constant for association (kass = 2,320,000 M(-1) s(-1)). Labeling of the active site is dose-dependent as observed using 6-cyclohexylamine-4-piperazinyl-1,3,5-triazine-2-carbonitrile, as competitive inhibitor of cathepsins. Finally we showed that Biot-(PEG)2-Ahx-LeuValGly-DMK may be a simple and convenient tool to label secreted and intracellular active cathepsins using a myelomonocytic cell line (THP-1 cells) as model.

Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

Oxidative C-H bond functionalization and ring expansion with TMSCHN2 : a copper(I)-catalyzed approach to dibenzoxepines and dibenzoazepines.

Tricyclic dibenzoxepines and dibenzazepines are important therapeutic agents for the pharmaceutical industry and academic research. However, their syntheses are generally rather tedious, requiring several steps that involve a Wagner-Meerwein-type rearrangement under harsh conditions. Herein, we present the first copper(I)-catalyzed oxidative CH bond functionalization and ring expansion with TMSCHN2 to yield these important derivatives in a facile and straightforward way.

Synthesis of Amathaspiramides by Aminocyanation of Enoates.

Concise routes for the total and formal syntheses of the amathaspiramides were developed through a formal [3+2] cycloaddition between lithium(trimethylsilyl)diazomethane and α,ß-unsaturated esters. The effectiveness of this new cycloaddition for the construction of Δ(2)-pyrazolines containing a α-tert-alkylamino carbon center and subsequent facile protonolytic N-N bond cleavage allows the synthesis of a key intermediate of the amathaspiramides and other α,α-disubstituted amino acid derivatives.

Photoaffinity labeling the propofol binding site in GLIC.

Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus . In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) Nature 469, 428-431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50 values of 20 and 50 µM, respectively. When [(3)H]AziPm (7 µM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziPm bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket.

Cysteine protease inhibitor 1 promotes metastasis by mediating an oxidative phosphorylation/MEK/ERK axis in esophageal squamous carcinoma cancer.

Cysteine protease inhibitor 1 (CST1) is a cystatin superfamily protein that inhibits cysteine protease activity and is reported to be involved in the development of many malignancies. Mitochondrial oxidative phosphorylation (OXPHOS) also plays an important role in cancer cell growth regulation. However, the relationship and roles of CST1 and OXPHOS in esophageal squamous cell carcinoma (ESCC) remains unclear. In our pilot study, CST1 was shown the potential of promoting ESCC migration and invasion by the activation of MEK/ERK pathway. Transcriptome sequencing analysis revealed that CST1 is closely associated with OXPHOS. Based on a real-time ATP rate assay, mitochondrial complex I enzyme activity assay, immunofluorescence, co-immunoprecipitation, and addition of the OXPHOS inhibitor Rotenone and MEK/ERK inhibitor PD98059, we determined that CST1 affects mitochondrial complex I enzyme activity by interacting with the GRIM19 protein to elevate OXPHOS levels, and a reciprocal regulatory relationship exists between OXPHOS and the MEK/ERK pathway in ESCC cells. Finally, an in vivo study demonstrated the potential of CST1 in ESCC metastasis through regulation of the OXPHOS and MEK/ERK pathways. This study is the first to reveal the oncogenic role of CST1 in ESCC development by enhancing mitochondrial respiratory chain complex I activity to activate the OXPHOS/MEK/ERK axis, and then promote ESCC metastasis, suggesting that CST1/OXPHOS is a promising target for ESCC treatment.

Diazirine-AIOC-NAADP, a Clickable-Photoactive NAADP Analog for Sea Urchin NAADP Binding Proteins.

Calcium ions (Ca2+) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca2+ in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive. Sea urchin NAADP binding proteins are important due to their unique identities and NAADP binding properties which may illuminate new signaling modalities in other species. Consequently, the development of new photoactive and clickable NAADP analogs with specificity for binding targets in sea urchin egg homogenates is a priority. We designed and synthesized diazirine-AIOC-NAADP, a photoactive and "clickable" NAADP analog, to specifically label and identify sea urchin NAADP receptors. This analog, synthesized using a chemo-enzymatic approach, induced Ca2+ release from sea urchin egg homogenates at low-micromolar concentrations. The ability of diazirine-AIOC-NAADP to mobilize Ca2+ in cultured human cells was investigated by microinjection of the probe into U2OS cells. Microinjected NAADP elicited a robust Ca2+ release, but even 6000-fold higher concentrations of diazirine-AIOC-NAADP were unable to release Ca2+. Our results indicate that our new probe is specifically recognized at low concentration by sea urchin egg NAADP receptors but not by the NAADP receptors in a human cultured cell line.