piSTING: A Pocket-Independent Agonist Based on Multivalency-Driven STING Oligomerization.

The stimulator of interferon genes (STING) pathway is a potent therapeutic target for innate immunity. Despite the efforts to develop pocket-dependent small-molecule STING agonists that mimic the endogenous STING ligand, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), most of these agonists showed disappointing results in clinical trials owing to the limitations of the STING pocket. In this study, we developed novel pocket-independent STING-activating agonists (piSTINGs), which act through multivalency-driven oligomerization to activate STING. Additionally, a piSTING-adjuvanted vaccine elicited a significant antibody response and inhibited tumour growth in therapeutic models. Moreover, a piSTING-based vaccine combination with aPD-1 showed remarkable potential to enhance the effectiveness of immune checkpoint blockade (ICB) immunotherapy. In particular, piSTING can strengthen the impact of STING pathway in immunotherapy and accelerate the clinical translation of STING agonists.

Isobaric Stable Isotope N-Phosphorylation Labeling (iSIPL) for Ultrasensitive Proteome Quantification.

Stable isotope chemical labeling methods have been widely used for high-throughput mass spectrometry (MS)-based quantitative proteomics in biological and clinical applications. However, the existing methods are far from meeting the requirements for high sensitivity detection. In the present study, a novel isobaric stable isotope N-phosphorylation labeling (iSIPL) strategy was developed for quantitative proteome analysis. The tryptic peptides were selectively labeled with iSIPL tag to generate the novel reporter ions containing phosphoramidate P-N bond with high intensities under lower collision energies. iSIPL strategy are suitable for peptide sequencing and quantitative analysis with high sensitivity and accuracy even for samples of limited quantity. Furthermore, iSIPL coupled with affinity purification and mass spectrometry was applied to measure the dynamics of cyclin dependent kinase 9 (CDK9) interactomes during transactivation of the HIV-1 provirus. The interaction of CDK9 with PARP13 was found to significantly decrease during Tat-induced activation of HIV-1 gene transcription, suggesting the effectiveness of iSIPL strategy in dynamic analysis of protein-protein interaction in vivo. More than that, the proposed iSIPL strategy would facilitate large-scale accurate quantitative proteomics by increasing multiplexing capability.

STING and TLR7/8 agonists-based nanovaccines for synergistic antitumor immune activation.

Immunostimulatory therapies based on pattern recognition receptors (PRRs) have emerged as an effective approach in the fight against cancer, with the ability to recruit tumor-specific lymphocytes in a low-immunogenicity tumor environment. The agonist cyclic dinucleotides (CDNs) of the stimulator of interferon gene (STING) are a group of very promising anticancer molecules that increase tumor immunogenicity by activating innate immunity. However, the tumor immune efficacy of CDNs is limited by several factors, including relatively narrow cytokine production, inefficient delivery to STING, and rapid clearance. In addition, a single adjuvant molecule is unable to elicit a broad cytokine response and thus cannot further amplify the anticancer effect. To address this problem, two or more agonist molecules are often used together to synergistically enhance immune efficacy. In this work, we found that a combination of the STING agonist CDGSF and the Toll-like receptor 7/8 (TLR7/8) agonist 522 produced a broader cytokine response. Subsequently, we developed multicomponent nanovaccines (MCNVs) consisting of a PC7A polymer as a nanocarrier encapsulating the antigen OVA and adjuvant molecules. These MCNVs activate bone marrow-derived dendritic cells (BMDCs) to produce multiple proinflammatory factors that promote antigen cross-presentation to stimulate specific antitumor T-cell responses. In in vivo experiments, we observed that MCNVs triggered a strong T-cell response in tumor-infiltrating lymphocytes, resulting in significant tumor regression and, notably, a 100% survival rate in mice through 25 days without other partnering therapies. These data suggest that our nanovaccines have great potential to advance cancer immunotherapy with increased durability and potency. Electronic Supplementary Material: Supplementary material (synthesis of CDGSF, 522, PC7A and OVA; preparation of MCNVs; representative gating strategies for flow cytometry) is available in the online version of this article at 10.1007/s12274-022-4282-x.

Recent Advances in Small Peptides of Marine Origin in Cancer Therapy.

Cancer is one of the leading causes of death in the world, and antineoplastic drug research continues to be a major field in medicine development. The marine milieu has thousands of biological species that are a valuable source of novel functional proteins and peptides, which have been used in the treatment of many diseases, including cancer. In contrast with proteins and polypeptides, small peptides (with a molecular weight of less than 1000 Da) have overwhelming advantages, such as preferential and fast absorption, which can decrease the burden on human gastrointestinal function. Besides, these peptides are only connected by a few peptide bonds, and their small molecular weight makes it easy to modify and synthesize them. Specifically, small peptides can deliver nutrients and drugs to cells and tissues in the body. These characteristics make them stand out in relation to targeted drug therapy. Nowadays, the anticancer mechanisms of the small marine peptides are still largely not well understood; however, several marine peptides have been applied in preclinical treatment. This paper highlights the anticancer linear and cyclic small peptides in marine resources and presents a review of peptides and the derivatives and their mechanisms.

Fully Synthetic Invariant NKT Cell-Dependent Self-Adjuvanting Antitumor Vaccines Eliciting Potent Immune Response in Mice.

Constructing an effective therapeutic cancer vaccine is very attractive and promising for cancer immunotherapy. However, the poor immunogenicity of tumor antigens and suppression of the immune system in the tumor microenvironment are two major obstacles for developing effective cancer vaccines. Invariant NKT cells (iNKT cells), which are essential bridges between the innate and adaptive immune systems, can be rapidly activated by their agonists and, consequently, evoke whole immune systems. Herein, we conjugated a potent agonist of the iNKT cell, α-galactosylceramide (α-GalCer), with the tumor-associated MUC1 glycopeptide antigens as novel self-adjuvanting cancer vaccines through click chemistry. Immunological studies revealed that the mouse immune system was potently evoked and that high levels of tumor-specific IgG antibodies were elicited by vaccine conjugates without an external adjuvant. The produced antibodies could specifically recognize and bind to antigen-expressing cancer cells and, subsequently, induce cytotoxicity through complement-dependent cytotoxicity. Thus, the insertion of α-GalCer significantly improved the immunogenicity of the MUC1 glycopeptide and induced strong antigen-specific antitumor responses, indicating that α-GalCer is an effective built-in adjuvant for constructing potent chemical synthetic antitumor vaccines.

Plasminogen activator, tissue type regulates germinal vesicle breakdown and cumulus expansion of bovine cumulus-oocyte complex in vitro†.

Plasminogen activator, tissue type (PLAT) and its inhibitor serpin family E member 1 (SERPINE1) cooperatively regulate PLAT activity in various reproductive processes. However, it is unknown whether this includes bovine oocyte maturation. We addressed this question in the present study by evaluating PLAT and SERPINE1 protein localization in immature cumulus-oocyte complexes (COCs), as well as PLAT mRNA and protein expression in cultured COCs after 0, 8, 16, and 24 h of in vitro maturation (IVM). We also examined the effects of PLAT and SERPINE1 on germinal vesicle breakdown (GVBD) and oocyte cyclic 3' 5' adenosine monophosphate (cAMP) levels, cumulus expansion index, and expansion-related gene expression in oocytes derived from bovine COCs cultured for 4, 8, and 12 h and in COCs cultured for 16 h. Both PLAT and SERPINE1 localized in cumulus cells but only the latter was detected in oocytes. PLAT and SERPINE1 transcript levels increased during IVM; however, from 8 to 16 h, the levels of PLAT remained stable whereas those of SERPINE1 increased, resulting in a decline in PLAT concentration. Additionally, PLAT delayed GVBD, increased oocyte cAMP levels, and blocked cumulus expansion and associated gene expression, which was reversed by SERPINE1 supplemented. Thus, PLAT delays bovine oocyte GVBD by enhancing oocyte cAMP levels during the first 8 h of IVM; suppression of PLAT activity via accumulation of SERPINE1 in COCs results in cumulus expansion from 8 to 16 h of IVM. These findings provide novel insights into the molecular mechanisms underlying in vitro bovine oocyte maturation.

Inhibition of K-Ras4B-plasma membrane association with a membrane microdomain-targeting peptide.

The association of K-Ras4B protein with plasma membrane (PM) is required for its signaling activity. Thus, direct inhibition of K-Ras4B-PM interaction could be a potential anti-Ras therapeutic strategy. However, it remains challenging to modulate such protein-PM interaction. Based on Ras isoform-specific PM microdomain localization patterns, we have developed a potent and isoform-selective peptide inhibitor, Memrasin, for detachment of K-Ras4B from the PM. Memrasin is one of the first direct inhibitors of K-Ras4B-PM interaction, and consists of a membrane ld region-binding sequence derived from the C-terminal region of K-Ras4B and an endosome-escape enhancing motif that can aggregate on membrane. It forms peptide-enriched domains in the ld region, abrogates the tethering of K-Ras4B to the PM and accordingly impairs Ras signaling activity, thereby efficiently decreasing the viability of several human lung cancer cells in a dose-responsive and K-Ras dependent manner. Memrasin provides a useful tool for exploring the biological function of K-Ras4B on or off the PM and a potential starting point for further development into anti-Ras therapeutics.

TDP-43 specific reduction induced by Di-hydrophobic tags conjugated peptides.

TAR DNA binding protein 43 (TDP-43) is a key target in amyotrophic lateral sclerosis (ALS) treatment. Here, based on hydrophobic tagging strategy, we designed and synthesized a series of single or double hydrophobic tags conjugated peptides D1-D8. Among them, it was found that D4 displayed strongest ability to induce TDP-43 degradation in cells. D4 could reduce TDP-43 induced cytotoxicity. Besides, D4 could reduce TDP-43 levels in a transgenic drosophila model.

Targeting STING with cyclic di-GMP greatly augmented immune responses of glycopeptide cancer vaccines.

Cyclic di-GMP (CDG) was applied to MUC1 glycopeptide-based cancer vaccines with physical mixing and built-in (at 2'-OH of CDG) strategies for activating the STING pathway. CDG in both strategies behaved as a potent immunostimulant and contributed to high titers of IgG antibodies and the expression of multiple cytokines.

De Novo Design To Synthesize Lanthipeptides Involving Cascade Cysteine Reactions: SapB Synthesis as an Example.

Lanthipeptides are a family of ribosomally synthesized peptides that have crucial biological functions. However, due to their complicated structures, the total synthesis of lanthipeptides is challenging. Here, a novel strategy to construct lanthipeptides is described, which involves cascade reactions of cysteine, including Cys disalkylation elimination, Michael reaction, and native chemical ligation. We utilized this strategy to synthesize lanthipeptide SapB as an example. This methodology has the potential to obtain lanthipeptides and their analogues for biological research and drug discovery.

Evolutionary relationships between seryl-histidine dipeptide and modern serine proteases from the analysis based on mass spectrometry and bioinformatics.

Seryl-histidine dipeptide (Ser-His) has been recognized as the shortest peptide with hydrolysis cleavage activity; however, its protein cleavage spectrum has not yet been fully explored. Here, four differently folded proteins were treated with Ser-His, and the digestion products were evaluated with high-resolution mass spectrometry. The cleavage efficiency and cleavage propensity of Ser-His against these protein substrates were calculated at both the primary and secondary sequence levels. The above experiments show that Ser-His cleaves a broad spectrum of substrate proteins of varying secondary structures. Moreover, Ser-His could cleave at all 20 amino acids with different efficiencies according to the protein, which means that Ser-His has the original digestion function of serine proteases. Furthermore, we collected and compared the catalytic sites and cleavage sites of 340 extant serine proteases derived from 17 representative organisms. A consensus motif Ser-[X]-His was identified as the major pattern at the catalytic sites of serine proteases from all of the organisms represented except Danio rerio, which uses Ser-Lys instead. This finding indicates that Ser-His is the core component of the serine protease catalytic site. Moreover, our analysis revealed that the cleavage sites of modern serine proteases have become more specific over the evolutionary history of this family. Based on the above analysis results, it could be found that Ser-His is likely the original serine protease and maybe the evolutionary core of modern serine proteases.

Self-Assembled Nano-Immunostimulant for Synergistic Immune Activation.

Immunotherapy has become one of the most promising therapies for the treatment of diseases. Synthetic immunostimulants and nanomaterial immunostimulant systems are indispensable for the activation of the immune system in cancer immunotherapy. Herein, a strategy for preparing self-assembled nano-immunostimulants (SANIs) for synergistic immune activation is reported. Three immunostimulants self-assemble into nanoparticles through electrostatic interactions. SANIs showed strong synergistic immunostimulation in macrophages. SANIs could also induce a strong antitumor immune response to inhibit tumor growth in mice and act as an efficient adjuvant of antitumor vaccines. Therefore, SANIs may be generally applied in cancer immunotherapy. This novel SANI strategy provides a new way for the development of both immunostimulants and -suppressants.

Semi-synthesis of murine prion protein by native chemical ligation and chemical activation for preparation of polypeptide-α-thioester.

Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three-dimensional structure domain was constructed from three segments murine PrP (mPrP)(90-177), mPrP(178-212), and mPrP(213-230) by combining protein expression, chemical synthesis and chemical ligation. The protein sequence 90 to 177 was obtained from expression and finally converted into the polypeptide hydrazide by chemical activation of a cysteine in the tail. The other two polypeptide fragments of the C-terminal were obtained by chemical synthesis, which utilized the strategies of isopeptide and pseudoproline building blocks to complete the synthesis of such difficult sequences. The three segments were finally assembled by sequentially using native chemical ligation. This strategy will allow more straightforward access to homogeneously modified PrP variants. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Phosphorylation Weakens but Does Not Inhibit Membrane Binding and Clustering of K-Ras4B.

K-Ras4B is one of the most frequently mutated Ras isoforms in cancer. The signaling activity of K-Ras4B depends on its localization to the plasma membrane (PM), which is mainly mediated by its polybasic farnesylated C-terminus. On top of the constitutive cycles that maintain the PM enrichment of K-Ras4B, conditional phosphorylation at Ser181 located within this motif has been found to be involved in regulating K-Ras4B's cell distribution and signaling activity. However, discordant observations have undermined our understanding of the role this phosphorylation plays. Here, we report an efficient strategy for producing K-Ras4B simultaneously bearing phosphate, farnesyl, and methyl modifications on a preparative scale, a very useful in vitro system when used in concert with model biomembranes. By using this system, we determined that phosphorylation at Ser181 does not fully inhibit membrane binding and clustering of K-Ras4B but reduces its membrane binding affinity, depending on membrane fluidity. In addition, phosphorylated K-Ras4B maintains tight association with its cytosolic shuttle protein PDEδ. After delivering K-Ras4B containing nonhydrolyzable phosphoserine mimetic into cells, the protein displayed a decreasing PM distribution compared with nonphosphorylable K-Ras4B, implying that phosphorylation might facilitate the dissociation of K-Ras4B from the PM. In addition, phosphorylation does not alter the localization of K-Ras4B in the liquid-disordered lipid subdomains of the membrane but slightly alters the thermotropic properties of K-Ras4B-incorporated membranes probably due to minor differences in membrane partitioning and dynamics. These results provide novel mechanistic insights into the role that phosphorylation at Ser181 plays in regulating K-Ras4B's distribution and activity.

Selective inhibition of cancer cells by enzyme-induced gain of function of phosphorylated melittin analogues.

The selective killing of cancer cells and the avoidance of drug resistance are still difficult challenges in cancer therapy. Here, we report a new strategy that uses enzyme-induced gain of function (EIGF) to regulate the structure and function of phosphorylated melittin analogues (MelAs). Original MelAs have the capacity to disrupt plasma membranes and induce cell death without selectivity. However, phosphorylation of Thr23 on one of the MelAs (MelA2-P) efficiently ameliorated the membrane lysis potency as well as the cytotoxicity for normal mammalian cells. After treatment with alkaline phosphatase (ALP), which is more active in cancer cells than normal cells, MelA2-P restored the pore-forming function around the cancer cells and induced cancer cell death selectively. This mechanism was independent of the receptor proteins and the cell uptake process, which may partially bypass the development of drug resistance in cancer cells.

Phosphorylation induces distinct alpha-synuclein strain formation.

Synucleinopathies are a group of neurodegenerative diseases associated with alpha-synuclein (α-Syn) aggregation. Recently, increasing evidence has demonstrated the existence of different structural characteristics or 'strains' of α-Syn, supporting the concept that synucleinopathies share several common features with prion diseases and possibly explaining how a single protein results in different clinical phenotypes within synucleinopathies. In earlier studies, the different strains were generated through the regulation of solution conditions, temperature, or repetitive seeded fibrillization in vitro. Here, we synthesize homogeneous α-Syn phosphorylated at serine 129 (pS129 α-Syn), which is highly associated with the pathological changes, and demonstrate that phosphorylation at Ser129 induces α-Syn to form a distinct strain with different structures, propagation properties, and higher cytotoxicity compared with the wild-type α-Syn. The results are the first demonstration that post-translational modification of α-Syn can induce different strain formation, offering a new mechanism for strain formation.

Synthetic MUC1 Antitumor Vaccine Candidates with Varied Glycosylation Pattern Bearing R/S-configured Pam3 CysSerLys4.

The Toll-like receptor 2 ligand Pam3 CysSer is of particular interest for the construction synthetic vaccines because of its ability to stimulate of the innate immune system. Such vaccines usually comprise Pam3 CysSer with the natural R-configuration at the glycerol 2-position. Pam3 CysSer peptide vaccines with natural configuration have been shown to be more efficient than the corresponding R/S diastereomers. In order to clarify whether the effect of the configuration of Pam3 Cys on the immune response also applies to glycopeptide vaccines, MUC1 glycopeptide-lipopeptide vaccines bearing either R- or R/S-configured Pam3 CysSerLys4 were compared for their immunological effects. In order to find out whether glycosylated MUC1 tandem repeat domains comprise not only B-cell epitopes but also T-cell epitopes, two-component vaccines containing the Pam3 CysSerLys4 lipopeptide and MUC1 glycopeptides with various glycosylation patterns were synthesized, and their immune reactions in mice were studied.

Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation.

Tau, an important pathological protein of Alzheimer's disease (AD), can mediate the toxicity of amyloid ß (Aß). Thus, reduction of Tau with chemical molecules may offer a novel strategy for treating AD. Here, we designed and synthesized a series of multifunctional molecules that contained Tau-recognition moieties and E3 ligase-binding moieties to enhance Tau degradation. Among these molecules, TH006 had the highest activity of inducing Tau degradation by increasing its poly-ubiquitination. The decrement in Tau induced by TH006 could decrease the cytotoxicity caused by Aß. Furthermore, TH006 could regulate the Tau level in the brain of an AD mouse model. Therefore, partial reduction of Tau with such multifunctional peptides may open up a novel therapeutic strategy for AD treatment.

Tracing the nitrogen metabolites of glycine using (15)N-glycine and mass spectrometry.

RATIONALE: Glycine is the smallest amino acid used in protein synthesis, but it is also a very important precursor for the biosynthesis of other nitrogen-containing metabolites, such as purine nucleosides and nucleotides for synthesis of RNA, DNA etc. Abnormalities in glycine metabolism therefore cause diseases such as cancer. A quick and unambiguous method to trace the metabolites arising from glycine is required for targeting defect points within metabolic networks. METHODS: This paper describes a method for using (15)N-glycine to culture A549 cancer cells for use with high-resolution mass spectrometry (HRMS) and tandem mass spectrometry (HRMS(2)) that can detect the (M+1)/M pair peaks appearing in the cell metabolites. The 1 Da difference in the pair peaks can be used to point out and identify the nitrogen metabolites of glycine. RESULTS: Thirteen nitrogen-containing metabolites derived from glycine were confirmed. Among them were metabolites containing purine, such as adenine, adenosine, AMP, ADP, ATP, S-adenosylmethionine and γ-glutathione; these were the most sensitive to the (15)N-glycine-enrichment technique. Therefore, they are promising biomarkers for monitoring the glycine metabolism network. CONCLUSIONS: The method developed here could be applied to investigations of metabolism of other amino acids, and for drug discovery studies targeting the enzymes related to amino acid metabolism.

Glycopeptide Nanoconjugates Based on Multilayer Self-Assembly as an Antitumor Vaccine.

Antitumor vaccine, which is promising for tumor therapy, has been extensively studied. Some encouraging results of chemically synthetic vaccine designs based on the tumor-associated antigen mucin 1 have been achieved. However, some shortcomings such as low efficiency and difficult purification restrict their clinical application. To overcome these difficulties, we designed a novel antitumor vaccine of glycopeptide nanoconjugates based on the multilayer self-assembly through the interaction of positive and negative charges. This vaccine formed the spherical structure and effectively activated the macrophage in vitro. Besides, it also induced high titer of antibodies against mucin 1 glycopeptide. The induced antibodies could highly bind to the tumor cells and effectively kill them by activation of the complement dependent cytotoxicity complex. This novel strategy provides a new way for the development of simple and effective antitumor vaccine.