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.

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.

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.

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.

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.

Photothermal Enhanced and Tumor Microenvironment Responsive Nanozyme for Amplified Cascade Enzyme Catalytic Therapy.

Nanocatalysts, a class of nanomaterials with intrinsic enzyme-like activities, have been widely investigated for cancer catalytic therapy in recent years. However, precise construction of nanocatalysts with excellent enzyme catalytic activity and biosafety for tumor therapy still remains challenging. Here, a biodegradable nanocatalyst, PEGylated Cux Mny Sz (PCMS), is reported that can promote cascade catalytic reactions in tumor microenvironment (TME) while confining off-target side effects on normal tissues. PCMS not only catalyzes the cascade conversion of endogenous hydrogen peroxide (H2 O2 ) to oxygen (O2 ) via catalase-like activity and then to superoxide radical (·O2 - ) via oxidase-like activity in the TME, but also effectively depletes intracellular glutathione via glutathione oxidase-like activity. The cascade catalytic reactions, by taking advantage of high H2 O2 level in tumor cells, result in an enhanced enzyme catalytic effect in generation of ·O2 - . More importantly, PCMS exhibits prominent photothermal effect under NIR-II 1064 nm laser irradiation that can further enhance chemodynamic therapy (CDT) efficacy in tumors. In addition, the biodegradation in TME and excellent photothermal effect of PCMS are beneficial to magnetic resonance imaging, photoacoustic imaging and infrared thermal imaging, resulting in tracing the fate of PCMS in vivo. This study provides a new tool for rational design of TME-responsive nanocatalysts with high biocompatibility for tumor catalytic therapy.

Structural basis for product specificities of MLL family methyltransferases.

Human mixed-lineage leukemia (MLL) family methyltransferases methylate histone H3 lysine 4 to different methylation states (me1/me2/me3) with distinct functional outputs, but the mechanism underlying the different product specificities of MLL proteins remains unclear. Here, we develop methodologies to quantitatively measure the methylation rate difference between mono-, di-, and tri-methylation steps and demonstrate that MLL proteins possess distinct product specificities in the context of the minimum MLL-RBBP5-ASH2L complex. Comparative structural analyses of MLL complexes by X-ray crystal structures, fluorine-19 nuclear magnetic resonance, and molecular dynamics simulations reveal that the dynamics of two conserved tyrosine residues at the "F/Y (phenylalanine/tyrosine) switch" positions fine-tune the product specificity. The variation in the intramolecular interaction between SET-N and SET-C affects the F/Y switch dynamics, thus determining the product specificities of MLL proteins. These results indicate a modified F/Y switch rule applicable for most SET domain methyltransferases and implicate the functional divergence of MLL proteins.

EPR-based in situ enzymatic activity detection of endogenous caspase-3 in apoptosis cell lysates.

Caspase-3 plays a vital role in cell apoptosis and related diseases. The detection and characterization of endogenous active caspase-3 are of immense value not only for mechanism studies of apoptosis but also for the diagnosis and treatment of apoptosis-related diseases. Here, an electron paramagnetic resonance (EPR)-based enzymatic assay was developed for the detection of caspase-3 activity both in vitro and in apoptosis cells. This assay uses a sandwich-like probe composed of a caspase-3-specific peptide segment (DEVD) conjugated to an EPR-detectable nitroxide spin label and magnetic beads (MBs). Cleavage of the "Nitroxide-Peptide-MBs" sandwich probe caspase-3 will release the nitroxide, which is readily detected by EPR after magnetic separation, resulting in a distinct EPR "off/on" transition. This assay takes advantage of the specific cleavage of DEVD-containing peptides by caspase-3 for high specificity, magnetic beads for fast magnetic separation, and EPR spectroscopy for considerably high detection sensitivity (LODs for caspase-3 are 116 nM at 60 min and 58 nM at 120 min). Importantly, the assay was proven to be compatible with complex biological samples and can detect the endogenous active caspase-3, thereby providing potential applications in the screening of protease-targeted drugs and the diagnosis of protease-associated diseases.

Structural insights into the activation of somatostatin receptor 2 by cyclic SST analogues.

The endogenous cyclic tetradecapeptide SST14 was reported to stimulate all five somatostatin receptors (SSTR1-5) for hormone release, neurotransmission, cell growth arrest and cancer suppression. Two SST14-derived short cyclic SST analogues (lanreotide or octreotide) with improved stability and longer lifetime were developed as drugs to preferentially activate SSTR2 and treat acromegalia and neuroendocrine tumors. Here, cryo-EM structures of the human SSTR2-Gi complex bound with SST14, octreotide or lanreotide were determined at resolutions of 2.85 Å, 2.97 Å, and 2.87 Å, respectively. Structural and functional analysis revealed that interactions between ß-turn residues in SST analogues and transmembrane SSTR2 residues in the ligand-binding pocket are crucial for receptor binding and functional stimulation of the two SST14-derived cyclic octapeptides. Additionally, Q1022.63, N2766.55, and F2947.35 could be responsible for the selectivity of lanreotide or octreotide for SSTR2 over SSTR1 or SSTR4. These results provide valuable insights into further rational development of SST analogue drugs targeting SSTR2.

Mechanistic investigation of B12-independent glycerol dehydratase and its activating enzyme GD-AE.

B12-Independent glycerol dehydratase (GD) is a glycyl radical enzyme in the biotransformation of glycerol to 1,3-propanediol. GD requires the activating enzyme GD-AE to initiate the radical reaction. GD-AE belongs to the radical S-adenosyl-L-methionine (SAM) enzyme superfamily. However, a previous study showed that GD-AE cleaves SAM unconventionally to generate 5'-deoxy-5'-methylthioadenosine. Herein, we show that GD-AE actually cleaves SAM to form 5'-deoxyadenosine, similar to other radical SAM enzymes. Furthermore, with the synthesized glycerol analogue 2-deoxy-2-fluoroglycerol, we demonstrate that B12-independent GD catalyzes the glycerol dehydration reaction by direct elimination of the C-2 hydroxyl group of a ketyl radical rather than 1,2-OH migration.

Helicobacter pylori FabX contains a [4Fe-4S] cluster essential for unsaturated fatty acid synthesis.

Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter pylori, a bacterium etiologically related to 95% of gastric cancers. Here, we present the crystal structures of FabX alone and in complexes with an octanoyl-acyl carrier protein (ACP) substrate or with holo-ACP. FabX belongs to the nitronate monooxygenase (NMO) flavoprotein family but contains an atypical [4Fe-4S] cluster absent in all other family members characterized to date. FabX binds ACP via its positively charged α7 helix that interacts with the negatively charged α2 and α3 helices of ACP. We demonstrate that the [4Fe-4S] cluster potentiates FMN oxidation during dehydrogenase catalysis, generating superoxide from an oxygen molecule that is locked in an oxyanion hole between the FMN and the active site residue His182. Both the [4Fe-4S] and FMN cofactors are essential for UFA synthesis, and the superoxide is subsequently excreted by H. pylori as a major resource of peroxide which may contribute to its pathogenic function in the corrosion of gastric mucosa.

A mirror-image protein-based information barcoding and storage technology.

A mirror-image protein-based information barcoding and storage technology wherein D-amino acids are used to encode information into mirror-image proteins that are chemically synthesized is described. These mirror-image proteins were then fused into various materials from which information-encoded objects were produced. Subsequently, the mirror-image proteins were extracted from the objects using biotin-streptavidin resin-mediated specific enrichment and cleaved using an Ni(II)-mediated selective peptide cleavage. Protein sequencing was accomplished using liquid chromatography/tandem mass spectrometry (LC-MS/MS) and then transcoded into the recorded information. We demonstrated the use of this technology to encode Chinese words into mirror-image proteins, which were then fused onto a poly(ethylene terephthalate) (PET) film and retrieved and decoded by LC-MS/MS sequencing. Compared to information barcoding and storage technologies using natural biopolymers, the mirror-image biopolymers used in our technology may be more stable and durable.

Structural insights into human acid-sensing ion channel 1a inhibition by snake toxin mambalgin1.

Acid-sensing ion channels (ASICs) are proton-gated cation channels that are involved in diverse neuronal processes including pain sensing. The peptide toxin Mambalgin1 (Mamba1) from black mamba snake venom can reversibly inhibit the conductance of ASICs, causing an analgesic effect. However, the detailed mechanism by which Mamba1 inhibits ASIC1s, especially how Mamba1 binding to the extracellular domain affects the conformational changes of the transmembrane domain of ASICs remains elusive. Here, we present single-particle cryo-EM structures of human ASIC1a (hASIC1a) and the hASIC1a-Mamba1 complex at resolutions of 3.56 and 3.90 Å, respectively. The structures revealed the inhibited conformation of hASIC1a upon Mamba1 binding. The combination of the structural and physiological data indicates that Mamba1 preferentially binds hASIC1a in a closed state and reduces the proton sensitivity of the channel, representing a closed-state trapping mechanism.

Chemical synthesis and biological activity of peptides incorporating an ether bridge as a surrogate for a disulfide bond.

Disulfide bridges contribute to the definition and rigidity of polypeptides, but they are inherently unstable in reducing environments and in the presence of isomerases and nucleophiles. Strategies to address these deficiencies, ideally without significantly perturbing the structure of the polypeptide, would be of great interest. One possible surrogate for the disulfide bridge is a simple thioether, but these are susceptible to oxidation. We report the introduction of an ether linkage into the biologically active, disulfide-rich peptides oxytocin, tachyplesin I, and conotoxin α-ImI, using an ether-containing diaminodiacid as the key building block, obtained by the stereoselective ring-opening addition reaction of an aziridine skeleton with a hydroxy group. NMR studies indicated that the derivatives with an ether surrogate bridge exhibited very small change of their three-dimensional structures. The analogs obtained using this novel substitution strategy were found to be more stable than the original peptide in oxidative and reductive conditions; without a loss of bioactivity. This strategy is therefore proposed as a practical and versatile solution to the stability problems associated with cysteine-rich peptides.

Chemical Synthesis of Native S-Palmitoylated Membrane Proteins through Removable-Backbone-Modification-Assisted Ser/Thr Ligation.

The preparation of native S-palmitoylated (S-palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S-palm membrane proteins by removable-backbone-modification-assisted Ser/Thr ligation (RBMGABA -assisted STL). This method involves two critical steps: 1) synthesis of S-palm peptides by a new γ-aminobutyric acid based RBM (RBMGABA ) strategy, and 2) ligation of the S-palm RBM-modified peptides to give the desired S-palm product by the STL method. The utility of the RBMGABA -assisted STL method was demonstrated by the synthesis of rabbit S-palm sarcolipin (SLN) and S-palm matrix-2 (M2) ion channel. The synthesis of S-palm membrane proteins highlights the importance of developing non-NCL methods for chemical protein synthesis.

Thiirane linkers directed histone H2A diubiquitination suggests plasticity in 53BP1 recognition.

Polyubiquitination with diverse linkages on histones provides another layer of accuracy and complexity for epigenetic regulation, which is rarely studied. Herein, K27 or K48-diubiquitin modified H2A analogues were chemically synthesized using thiirane linkers. These permitted in vitro binding studies suggested the plasticity of ubiquitin chains in 53BP1 recognition.