Imaging and quantification of neuropeptides in mouse pituitary tissue by atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

RATIONALE: Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry has enabled the untargeted analysis and imaging of neuropeptides and proteins in biological tissues under ambient conditions. Sensitivity in AP-MALDI can be improved by using sample-specific preparation methods. METHODS: A comprehensive and detailed optimization strategy including instrument parameters, matrix spraying and sample tissue washing pretreatment was implemented to enhance the sensitivity and coverage of neuropeptides in mouse pituitary tissues by commercial AP-MALDI mass spectrometry imaging (MSI). RESULTS: The sensitivity of a commercial AP-MALDI system for endogenous neuropeptides in mouse pituitary was enhanced by up to 15.2-fold by shortening the transmission gap from the sample plate to the inlet, attaching copper adhesive tape to an indium tin oxide-coated glass slide, optimizing the matrix spray solvent and using sample tissue washing pretreatment. Following careful optimization, the distributions of nine endogenous neuropeptides were successfully visualized in the pituitary. Furthermore, the quantitative capability of AP-MALDI for neuropeptides was evaluated and the concentrations of neuropeptides oxytocin and vasopressin in the pituitary posterior lobe were increased approximately twofold under hypertonic saline stress. CONCLUSION: Mouse pituitary neuropeptides have emerged as important signaling molecules due to their role in stress response. This work indicates the potential of modified AP-MALDI as a promising AP MSI method for in situ visualization and quantification of neuropeptides in complex biological tissues.

Development of Transmission Ambient Pressure Laser Desorption Ionization/Postphotoionization Mass Spectrometry Imaging.

Laser-based high-resolution mass spectrometry imaging at ambient conditions has promising applications in life science. However, the ion yield during laser desorption/ablation is poor. Here, transmission atmospheric pressure laser desorption ionization combined with a compact postphotoionization (t-AP-LDI/PI) assembly with a krypton discharge lamp was developed for the untargeted imaging of various biomolecules. The spatial distributions of numerous lipid classes, fatty acids, neurotransmitters, and amino acids in the subregions of mouse cerebellum tissue were obtained. Compared with single laser ablation, the sensitivities for most analytes were increased by 1 to 3 orders of magnitude by dopant-assisted postphotoionization. After careful optimization, a spatial resolution of 4 µm could be achieved for the metabolites in mouse hippocampus tissue. Finally, the melanoma tissue slices were analyzed using t-AP-LDI/PI MSI, which revealed the metabolic heterogeneity of the melanoma microenvironment and exhibited the phenomenon of abnormal proliferation and invasion trends in tumor cells.

Peptide Stapling by Crosslinking Two Amines with α-Ketoaldehydes through Diverse Modified Glyoxal-Lysine Dimer Linkers.

α-Ketoaldehydes play versatile roles in the ubiquitous natural processes of protein glycation. However, leveraging the reactivity of α-ketoaldehydes for biomedical applications has been challenging. Previously, the reactivity of α-ketoaldehydes with guanidine has been harnessed to design probes for labeling Arg residues on proteins in an aqueous medium. Herein, a highly effective, broadly applicable, and operationally simple protocol for stapling native peptides by crosslinking two amino groups through diverse imidazolium linkers with various α-ketoaldehyde reagents is described. The use of hexafluoroisopropanol as a solvent facilitates rapid and clean reactions under mild conditions and enables unique selectivity for Lys over Arg. The naturally occurring GOLD/MOLD linkers have been expanded to encompass a wide range of modified glyoxal-lysine dimer (OLD) linkers. In a proof-of-concept trial, these modular stapling reactions enabled a convenient two-round strategy to streamline the structure-activity relationship (SAR) study of the wasp venom peptide anoplin, leading to enhanced biological activities.

De Novo Screening and Mirror Image Isomerization of Linear Peptides Targeting α7 Nicotinic Acetylcholine Receptor.

As ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs) are widely distributed in the central and peripheral nervous systems and are associated with the pathogenesis of various degenerative neurological diseases. Here, we report the results of phage display-based de novo screening of an 11-residue linear peptide (named LKP1794) that targets the α7 nAChR, which is among the most abundant nAChR subtypes in the brain. Moreover, two d-peptides were generated through mirror image and/or primary sequence inverso isomerization (termed DRKP1794 and DKP1794) and displayed improved inhibitory effects (IC50 = 0.86 and 0.35 µM, respectively) on α7 nAChR compared with the parent l-peptide LKP1794 (IC50 = 2.48 µM), which markedly enhanced serum stability. A peptide-based fluorescence probe was developed using proteolytically resistant DKP1794 to specifically image the α7 nAChR in living cells. This work provides a new peptide tool to achieve inhibitory modulation and specifically image the α7 nAChR.

Cryo-EM structure of HQNO-bound Alternative Complex III from the anoxygenic phototrophic bacterium Chloroflexus aurantiacus.

Alternative complex III (ACIII) couples quinol oxidation and electron acceptor reduction with potential transmembrane proton translocation. It is compositionally and structurally different from the cytochrome bc1/b6f complexes, but functionally replaces these enzymes in the photosynthetic and/or respiratory electron transport chains (ETCs) of many bacteria. However, the true compositions and architectures of ACIIIs remain unclear, as do their structural and functional relevance in mediating the ETCs. We here determined cryogenic electron microscopy structures of photosynthetic ACIII isolated from Chloroflexus aurantiacus (CaACIIIp), in apo-form and in complexed form bound to a menadiol analog 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Besides six canonical subunits (ActABCDEF), the structures revealed conformations of two previously unresolved subunits, ActG and I, which contributed to the complex stability. We also elucidated the structural basis of menaquinol oxidation and subsequent electron transfer along the [3Fe-4S]-6 hemes wire to its periplasmic electron acceptors, using electron paramagnetic resonance (EPR), spectroelectrochemistry, enzymatic analyses and molecular dynamics (MD) simulations. A unique insertion loop in ActE was shown to function in determining the binding specificity of CaACIIIp for downstream electron acceptors. This study broadens our understanding of the structural diversity and molecular evolution of ACIIIs, enabling further investigation of the (mena)quinol oxidoreductases evolved coupling mechanism in bacterial energy conservation.

A cytochrome c551 mediates the cyclic electron transport chain of the anoxygenic phototrophic bacterium Roseiflexus castenholzii.

Roseiflexus castenholzii is a gram-negative filamentous phototrophic bacterium that carries out anoxygenic photosynthesis through a cyclic electron transport chain (ETC). The ETC is composed of a reaction center (RC)-light-harvesting (LH) complex (rcRC-LH); an alternative complex III (rcACIII), which functionally replaces the cytochrome bc1/b6f complex; and the periplasmic electron acceptor auracyanin (rcAc). Although compositionally and structurally different from the bc1/b6f complex, rcACIII plays similar essential roles in oxidizing menaquinol and transferring electrons to the rcAc. However, rcACIII-mediated electron transfer (which includes both an intraprotein route and a downstream route) has not been clearly elucidated, nor have the details of cyclic ETC. Here, we identify a previously unknown monoheme cytochrome c (cyt c551) as a novel periplasmic electron acceptor of rcACIII. It reduces the light-excited rcRC-LH to complete a cyclic ETC. We also reveal the molecular mechanisms involved in the ETC using electron paramagnetic resonance (EPR), spectroelectrochemistry, and enzymatic and structural analyses. We find that electrons released from rcACIII-oxidized menaquinol are transferred to two alternative periplasmic electron acceptors (rcAc and cyt c551), which eventually reduce the rcRC to form the complete cyclic ETC. This work serves as a foundation for further studies of ACIII-mediated electron transfer in anoxygenic photosynthesis and broadens our understanding of the diversity and molecular evolution of prokaryotic ETCs.

A light-driven enzymatic enantioselective radical acylation.

Enzymes are recognized as exceptional catalysts for achieving high stereoselectivities1-3, but their ability to control the reactivity and stereoinduction of free radicals lags behind that of chemical catalysts4. Thiamine diphosphate (ThDP)-dependent enzymes5 are well-characterized systems that inspired the development of N-heterocyclic carbenes (NHCs)6-8 but have not yet been proved viable in asymmetric radical transformations. There is a lack of a biocompatible and general radical-generation mechanism, as nature prefers to avoid radicals that may be harmful to biological systems9. Here we repurpose a ThDP-dependent lyase as a stereoselective radical acyl transferase (RAT) through protein engineering and combination with organophotoredox catalysis10. Enzyme-bound ThDP-derived ketyl radicals are selectively generated through single-electron oxidation by a photoexcited organic dye and then cross-coupled with prochiral alkyl radicals with high enantioselectivity. Diverse chiral ketones are prepared from aldehydes and redox-active esters (35 examples, up to 97% enantiomeric excess (e.e.)) by this method. Mechanistic studies reveal that this previously elusive dual-enzyme catalysis/photocatalysis directs radicals with the unique ThDP cofactor and evolvable active site. This work not only expands the repertoire of biocatalysis but also provides a unique strategy for controlling radicals with enzymes, complementing existing chemical tools.

In vivo labeling and quantitative imaging of neuronal populations using MRI.

The study of neural circuits, which underlies perception, cognition, emotion, and behavior, is essential for understanding the mammalian brain, a complex organ consisting of billions of neurons. To study the structure and function of the brain, in vivo neuronal labeling and imaging techniques are crucial as they provide true physiological information that ex vivo methods cannot offer. In this paper, we present a new strategy for in vivo neuronal labeling and quantification using MRI. We demonstrate the efficacy of this method by delivering the oatp1a1 gene to the target neurons using rAAV2-retro virus. OATP1A1 protein expression on the neuronal membrane increased the uptake of a specific MRI contrast agent (Gd-EOB-DTPA), leading to hyperintense signals on T1W images of labeled neuronal populations. We also used dynamic contrast enhancement-based methods to obtain quantitative information on labeled neuronal populations in vivo.

Fluoride permeation mechanism of the Fluc channel in liposomes revealed by solid-state NMR.

Solid-state nuclear magnetic resonance (ssNMR) methods can probe the motions of membrane proteins in liposomes at the atomic level and propel the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. In this work, we report our study on the fluoride channel Fluc-Ec1 in phospholipid bilayers based on ssNMR and molecular dynamics simulations. Previously unidentified fluoride binding sites in the aqueous vestibules were experimentally verified by 19F-detected ssNMR. One of the two fluoride binding sites in the polar track was identified as a water molecule by 1H-detected ssNMR. Meanwhile, a dynamic hotspot at loop 1 was observed by comparing the spectra of wild-type Fluc-Ec1 in variant buffer conditions or with its mutants. Therefore, we propose that fluoride conduction in the Fluc channel occurs via a "water-mediated knock-on" permeation mechanism and that loop 1 is a key molecular determinant for channel gating.

Modification of Low-Energy Surfaces Using Bicyclic Peptides Discovered by Phage Display.

Solid-binding peptides are a simple and versatile tool for the non-covalent modification of solid material surfaces, and a variety of peptides have been developed by reference to natural proteins or de novo design. Here, for the first time, we report the discovery of a bicyclic peptide targeting the heterogeneous material polypropylene by combining phage display technology and next-generation sequencing. We find that the enrichment properties of bicyclic peptides capable of binding to polypropylene are distinct from linear peptides, as reflected in amino acid abundance and a trend toward negative net charges and high hydrophobicity. The selected bicyclic peptide has a higher binding affinity for polypropylene compared with a previously reported linear peptide, enabling the hydrophilic and adhesive properties of the polypropylene to be more effectively enhanced. Our work paves the way for the exploration and utilization of conformational-restricted cyclic peptides as a new family of functionally evolvable agents for material surface modification.

Structural insights into neurokinin 3 receptor activation by endogenous and analogue peptide agonists.

Neurokinin 3 receptor (NK3R) is a tachykinin receptor essential for the hypothalamic-pituitary-gonadal axis. The endogenous peptide agonist neurokinin B (NKB) preferentially activates NK3R, while substance P (SP) binds preferentially to NK1R. In addition, the SP analogue senktide more potently activates NK3R than NKB and SP. However, the mechanisms of preferential binding of peptide and NK3R activation remain elusive. Herein, we determined the cryogenic electron microscopy (cryo-EM) structures of the NK3R-Gq complex bound to NKB, SP and senktide. The three NK3R-Gq/peptide complexes utilize a class of noncanonical receptor activation mechanisms. Combining the structural analysis and functional assay illustrated that the consensus C-termini of the three peptide agonists share a conserved binding mode to NK3R, while the divergent N-termini of the peptides confer the preferential binding of the agonist to NK3R. In addition, the specific interactions between the N-terminus of senktide and the N-terminus and extracellular loops (ECL2 and ECL3) of NK3R lead to the improved activation displayed by senktide compared to SP and NKB. These findings pave the way to understand tachykinin receptor subtype selectivity and provide ideas to rationally develop drugs targeting NK3R.

Cobalt-Catalyzed Facial-Selective Hydroalkylation of Cyclopropenes.

Cyclopropene hydrofunctionalization has been a promising strategy for accessing multi-substituted cyclopropanes; however, cyclopropene hydroalkylation remains underdeveloped. Herein, we report a low-valent CoH-catalyzed facial-selective cyclopropene hydroalkylation to access multi-substituted cyclopropanes. This reaction exhibits a broad substrate scope of alkyl halides and cyclopropenes and tolerates many functional groups. Moderate-to-good facial-selectivity is obtained without any directing groups. Mechanism studies provide evidence that alkyl radicals are generated from alkyl halides and irreversible CoH insertion is responsible for the facial-selectivity. Our preliminary exploration demonstrates that asymmetric cyclopropene hydroalkylation can be realized without conspicuous auxiliary groups.

IgG Fc-binding protein positively regulates the assembly of pore-forming protein complex ßγ-CAT evolved to drive cell vesicular delivery and transport.

Cell membranes form barriers for molecule exchange between the cytosol and the extracellular environments. ßγ-CAT, a complex of pore-forming protein BmALP1 (two ßγ-crystallin domains with an aerolysin pore-forming domain) and the trefoil factor BmTFF3, has been identified in toad Bombina maxima. It plays pivotal roles, via inducing channel formation in various intracellular or extracellular vesicles, as well as in nutrient acquisition, maintaining water balance, and antigen presentation. Thus, such a protein machine should be tightly regulated. Indeed, BmALP3 (a paralog of BmALP1) oxidizes BmALP1 to form a water-soluble polymer, leading to dissociation of the ßγ-CAT complex and loss of biological activity. Here, we found that the B. maxima IgG Fc-binding protein (FCGBP), a well-conserved vertebrate mucin-like protein with unknown functions, acted as a positive regulator for ßγ-CAT complex assembly. The interactions among FCGBP, BmALP1, and BmTFF3 were revealed by co-immunoprecipitation assays. Interestingly, FCGBP reversed the inhibitory effect of BmALP3 on the ßγ-CAT complex. Furthermore, FCGBP reduced BmALP1 polymers and facilitated the assembly of ßγ-CAT with the biological pore-forming activity in the presence of BmTFF3. Our findings define the role of FCGBP in mediating the assembly of a pore-forming protein machine evolved to drive cell vesicular delivery and transport.

Structure of the human respiratory complex II.

Human complex II is a key protein complex that links two essential energy-producing processes: the tricarboxylic acid cycle and oxidative phosphorylation. Deficiencies due to mutagenesis have been shown to cause mitochondrial disease and some types of cancers. However, the structure of this complex is yet to be resolved, hindering a comprehensive understanding of the functional aspects of this molecular machine. Here, we have determined the structure of human complex II in the presence of ubiquinone at 2.86 Å resolution by cryoelectron microscopy, showing it comprises two water-soluble subunits, SDHA and SDHB, and two membrane-spanning subunits, SDHC and SDHD. This structure allows us to propose a route for electron transfer. In addition, clinically relevant mutations are mapped onto the structure. This mapping provides a molecular understanding to explain why these variants have the potential to produce disease.

Total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates using side-chain Boc/tBu protecting groups.

A practical strategy for the total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates was developed. In this strategy, the Boc/tBu protecting groups are utilized for the side chains of Trp, His, Arg, Asp, and Glu, and is deprotected in borate buffer at 90 °C to avoid depurination of the oligonucleotide caused by strong acid treatment. The advantage of this strategy is that the abovementioned amino acids are readily available in the market and the side reaction of deguanidination of the Arg residue can be avoided. This side-chain Boc/tBu protection strategy will expand the applicability of total stepwise synthesis in the preparation of peptide-oligonucleotide conjugates.

Intracellular Construction of Cathepsin B-Guided Gadolinium Nanoparticles for Enhanced T2 -Weighted MR Tumor Imaging.

Magnetic resonance imaging (MRI) is a superior and noninvasive imaging technique with unlimited tissue penetration depth and superb spatiotemporal resolution, however, using intracellular self-assembly of Gd-containing nanoparticles to enhance the T2 -weighted MR contrast of cancer cells in vivo for precise tumor MRI is rarely reported. The lysosomal cysteine protease cathepsin B (CTSB) is regarded as an attractive biomarker for the early diagnosis of cancers and metastasis. Herein, taking advantage of a biocompatible condensation reaction, a "smart" Gd-based CTSB-responsive small molecular contrast agent VC-Gd-CBT is developed, which can self-assemble into large intracellular Gd-containing nanoparticles by glutathione reduction and CTSB cleavage to enhance the T2 -weighted MR contrast of CTSB-overexpressing MDA-MB-231 cells at 9.4 T. In vivo T2 -weighted MRI studies using MDA-MB-231 murine xenografts show that the T2 -weighted MR contrast change of tumors in VC-Gd-CBT-injected mice is distinctly larger than the mice injected with the commercial agent gadopentetate dimeglumine, or co-injected with CTSB inhibitor and VC-Gd-CBT, indicating that the accumulation of self-assembled Gd-containing nanoparticles at tumor sites effectively enhances the T2 -weighted MR tumor imaging. Hence, this CTSB-targeted small molecule VC-Gd-CBT has the potential to be employed as a T2 contrast agent for the clinical diagnosis of cancers at an early stage.

New IMB16-4 Hot-Melt Extrusion Preparation Improved Oral Bioavailability and Enhanced Anti-Cholestatic Effect on Rats.

Background: Cholestasis is challenging to treat due to lacked effective drugs. N-(3,4,5-trichlorophenyl)-2 (3-nitrobenzenesulfonamido) benzamide, abbreviated as IMB16-4, which may be effective for the treatment of cholestasis. However, its poor solubility and bioavailability seriously obstruct the research programs. Methods: A hot-melt extrusion (HME) preparation was first applied to increase the bioavailability of IMB16-4, the oral bioavailability, anti-cholestatic effect and vitro cytotoxicity of IMB16-4 and IMB16-4-HME were evaluated. Meanwhile, the molecular docking and qRT-PCR were used to validate the mechanism behind. Results: The oral bioavailability of IMB16-4-HME improved 65-fold compared with that of pure IMB16-4. Pharmacodynamics results demonstrated that IMB16-4-HME prominently decreased the serum levels of total bile acid (TBA) and alkaline phosphatase (ALP), but elevated the level of total bilirubin (TBIL) and direct bilirubin (DBIL). Histopathology revealed that IMB16-4-HME at lower dose exhibited stronger anti-cholestatic effect compared with pure IMB16-4. In addition, molecular docking demonstrated that IMB16-4 has great affinity with PPARα, and qRT-PCR results revealed that IMB16-4-HME significantly elevated the mRNA expression level of PPARα, but decreased the mRNA level of CYP7A1. Cytotoxicity assays demonstrated that the hepatotoxicity of IMB16-4-HME was absolutely attributed to IMB16-4, and the excipients of IMB16-4-HME may increase the drug load within HepG2 cells. Conclusion: The HME preparation significantly increased the oral bioavailability and anti-cholestatic effect of pure IMB16-4, but caused liver injury at high dose, which require a dose balance between the curative effect and safety in the future research.

Basis of the H2AK119 specificity of the Polycomb repressive deubiquitinase.

Repression of gene expression by protein complexes of the Polycomb group is a fundamental mechanism that governs embryonic development and cell-type specification1-3. The Polycomb repressive deubiquitinase (PR-DUB) complex removes the ubiquitin moiety from monoubiquitinated histone H2A K119 (H2AK119ub1) on the nucleosome4, counteracting the ubiquitin E3 ligase activity of Polycomb repressive complex 1 (PRC1)5 to facilitate the correct silencing of genes by Polycomb proteins and safeguard active genes from inadvertent silencing by PRC1 (refs. 6-9). The intricate biological function of PR-DUB requires accurate targeting of H2AK119ub1, but PR-DUB can deubiquitinate monoubiquitinated free histones and peptide substrates indiscriminately; the basis for its exquisite nucleosome-dependent substrate specificity therefore remains unclear. Here we report the cryo-electron microscopy structure of human PR-DUB, composed of BAP1 and ASXL1, in complex with the chromatosome. We find that ASXL1 directs the binding of the positively charged C-terminal extension of BAP1 to nucleosomal DNA and histones H3-H4 near the dyad, an addition to its role in forming the ubiquitin-binding cleft. Furthermore, a conserved loop segment of the catalytic domain of BAP1 is situated near the H2A-H2B acidic patch. This distinct nucleosome-binding mode displaces the C-terminal tail of H2A from the nucleosome surface, and endows PR-DUB with the specificity for H2AK119ub1.