Enzyme-Instructed Photoactivatable Supramolecular Antigens on Cancer Cell Membranes for Precision-Controlled T-Cell-Based Cancer Immunotherapy.

Cancer immunotherapies based on cytotoxic CD8+ T lymphocytes (CTLs) are highly promising for cancer treatment. The specific interaction between T-cell receptors and peptide-MHC-I complexes (pMHC-I) on cancer cell membranes critically determines their therapeutic outcomes. However, the lack of appropriate endogenous antigens for MHC-I presentation disables tumor recognition by CTLs. By devising three antigen-loaded self-assembling peptides of pY-K(Ag)-ERGD, pY-K(Ag)-E, and Y-K(Ag)-ERGD to noncovalently generate light-activatable supramolecular antigens at tumor sites in different manners, we report pY-K(Ag)-ERGD as a promising candidate to endow tumor cells with pMHC-I targets on demand. Specifically, pY-K(Ag)-ERGD first generates low-antigenic supramolecular antigens on cancer cell membranes, and a successive light pulse allows antigen payloads to efficiently release from the supramolecular scaffold, directly producing antigenic pMHC-I. Intravenous administration of pY-K(Ag)-ERGD enables light-controlled tumor inhibition when combined with adoptively transferred antigen-specific CTLs. Our strategy is feasible for broadening tumor antigen repertoires for T-cell immunotherapies and advancing precision-controlled T-cell immunotherapies.

In Situ Construction of Ferrocene-Containing Membrane-Bound Nanofibers for the Redox Control of Cancer Cell Death and Cancer Therapy.

Precise manipulation of cancer cell death by harnessing reactive oxygen species (ROS) is a promising strategy to defeat malignant tumors. However, it is quite difficult to produce active ROS with spatial precision and regulate their biological outcomes. We succeed here in selectively generating short-lived and lipid-reactive hydroxyl radicals (•OH) adjacent to cancer cell membranes, successively eliciting lipid peroxidation and ferroptosis. DiFc-K-pY, a phosphorylated self-assembling precursor that consists of two branched Fc moieties and interacts specifically with epidermal growth factor receptor, can in situ produce membrane-bound nanofibers and enrich ferrocene moieties on cancer cell membranes in response to alkaline phosphatase. Within the acidic tumor microenvironment, DiFc-K-pY nanofibers efficiently convert tumoral H2O2 to active •OH around the target cell membranes via Fenton-like reactions, leading to lipid peroxidation and ferroptosis with good cellular selectivity. Our strategy successfully prevents tumor progression with acceptable biocompatibility through intratumoral administration.

A Cascade-Targeted Enzyme-Instructed Peptide Self-Assembly Strategy for Cancer Immunotherapy through Boosting Immunogenic Cell Death.

Immunogenic cell death (ICD) approaches by encumbering mitochondrial functions provide great promise for the treatment of malignant tumors, but these kinds of ICD strategies are still in their infancy. Here, one multifunctional drug-loaded, cascade-targeted, and enzyme-instructed self-assembling peptide nanomedicine (Comp. 4) for ICD-based cancer therapy is constructed. Comp. 4 consists of 1) lonidamine (LND) that specifically interferes with mitochondrial functions; 2) a programmed death ligand 1 (PD-L1) binding peptide sequence (NTYYEDQG) and a mitochondria-specific motif (triphenylphosphonium, TPP) that can sequentially control the cell membrane and mitochondria targeting capacities, respectively; and 3) a -GD FD FpD Y- assembly core to in situ organize peptide assemblies responsive to alkaline phosphatase (ALP). Comp. 4 demonstrates noticeable structural and morphological transformations in the presence of ALP and produces peptide assemblies in mouse colon cancer cells (CT26) with high expressions of both ALP and PD-L1. Moreover, the presence of PD-L1- and mitochondria-specific motifs can assist Comp. 4 for effective endocytosis and endosomal escape, forming peptide assemblies and delivering LND into mitochondria. Consequently, Comp. 4 shows superior capacities to in vivo induce abundant mitochondrial oxidative stress, provoke robust ICD responses, and produce an immunogenic tumor microenvironment, successfully inhibiting CT26 tumor growth by eliciting a systemic ICD-based antitumor immunity.

Enzyme-Instructed Peptide Assembly Favored by Preorganization for Cancer Cell Membrane Engineering.

Innovative methods for engineering cancer cell membranes promise to manipulate cell-cell interactions and boost cell-based cancer therapeutics. Here, we illustrate an in situ approach to selectively modify cancer cell membranes by employing an enzyme-instructed peptide self-assembly (EISA) strategy. Using three phosphopeptides (pY1, pY2, and pY3) targeting the membrane-bound epidermal growth factor receptor (EGFR) and differing in just one phosphorylated tyrosine, we reveal that site-specific phosphorylation patterns in pY1, pY2, and pY3 can distinctly command their preorganization levels, self-assembling kinetics, and spatial distributions of the resultant peptide assemblies in cellulo. Overall, pY1 is the most capable of producing preorganized assemblies and shows the fastest dephosphorylation reaction in the presence of alkaline phosphatase (ALP), as well as the highest binding affinity for EGFR after dephosphorylation. Consequently, pY1 exhibits the greatest capacity to construct stable peptide assemblies on cancer cell membranes with the assistance of both ALP and EGFR. We further use peptide-protein and peptide-peptide co-assembly strategies to apply two types of antigens, namely ovalbumin (OVA) protein and dinitrophenyl (DNP) hapten respectively, on cancer cell membranes. This study demonstrates a very useful technique for the in situ construction of membrane-bound peptide assemblies around cancer cells and implies a versatile strategy to artificially enrich cancer cell membrane components for potential cancer immunotherapy.

Enzyme-instructed self-assembly enabled fluorescence light-up for alkaline phosphatase detection.

Alkaline phosphatase (ALP) exists in both normal and pathological tissues. Spatiotemporal variations in ALP levels can reveal its potential physiological functions and changes that occur during pathological conditions. However, it is still challenging to exploit fluorescent probes that can measure ALP activity under good spatial and temporal resolutions. Herein, enzyme-instructed self-assembly (EISA) was used to construct a high-performing analytical tool (MN-pY) to probe ALP activity. MN-pY alone (free state) showed negligible fluorescence but presented an almost 13-fold increase in fluorescence intensity in the presence of ALP (assembly state). Mechanism study indicated the increase in fluorescence intensity was due to hydrogelation and formation of supramolecular fibrils, mainly consisting of dephosphorylated MN-Y. The dephosphorylation and further fibrillation of MN-pY could induce the formation of a "hydrophobic pocket", leading to a further increase in fluorescence intensity. Moreover, MN-pY could selectively illuminate HeLa cells with a higher ALP expression but not LO2 cells with lower ALP levels, promising a potential application in cancer diagnosis.

Cross-Seeded Fibrillation Induced by Pyroglutamate-3 and Truncated Aß40 Variants Leads to Aß40 Structural Polymorphism Modulation and Elevated Toxicity.

The pathological amyloid plaques in Alzheimer's disease (AD) patients contain not only the wild-type ß-amyloid (wt-Aß) peptide sequences but also a variety of post-translationally modified variants. The pyroglutamate-3 Aß (pyroE3-Aß), which is generated from its truncated precursors ΔE3-Aß, shows the highest abundance among all modified Aß variants. Previous works have shown that pyroE3-Aß and/or ΔE3-Aß, compared with the wild-type sequences, led to a more rapid fibrillation process and final fibrils with higher neuronal cytotoxicity levels. However, much less is known about how the formation of pyroE3/ΔE3-Aß fibrils would affect the amyloid deposition of wt-Aß peptides, which are the main pathological events in AD. We show in the present work that the pyroE3/ΔE3-Aß40 fibrils differ significantly from the wt-Aß40 fibrils in terms of their molecular structures. When added into monomeric wt-Aß40 peptides, these variant fibrils can cross-seed the formation of wt-Aß40 fibrils with fibrillation kinetics that are greater than the self-seeded fibrillation of wt-Aß40. Furthermore, the cross-seeding process modulates the molecular structures of the yielded wt-Aß40 fibrils, which show similar features as their variant seeds. The cross-seeded fibrillation process also induces higher cytotoxicity levels compared with the self-seeded fibrillation of wt-Aß40. Overall, our results support the hypothesis that pyroE3 and ΔE3-Aß40 variants may serve as triggering factors of the pathological amyloid aggregation of wt-Aß40 and may underlie the pathological significance of pyroE3/ΔE3-Aß40 variants on the structural polymorphism of Aß deposits.

N-Terminal Modified Aß Variants Enable Modulations to the Structures and Cytotoxicity Levels of Wild-Type Aß Fibrils through Cross-Seeding.

Post-translational modifications (PTMs) of ß-amyloid (Aß) peptides are considered as triggering factors in sporadic Alzheimer's disease. However, studies to show the influence of pre-existing PTM-Aß fibrils on wild-type Aß peptides, which directly mimic the triggering scenarios, are rare. Here we show that three types of pathologically relevant PTM-Aß variants with modifications in a particular segment (from D7 to V12) of the primary sequence lead to distinct impacts on the fibrillization of wild-type Aß peptides. In general, the triggering effects are observed through cross-seeding between the PTM-Aß seeds and wild-type peptides, which consequently induce modulations in the resultant wild-type fibril structures and elevations in the fibrillar cytotoxicity levels. Modifications with a similar chemical nature, such as the S8-phosphorylation and Y10-nitration, both of which introduce additional side-chain negative charges, show comparable structural-modulation and cytotoxicity-elevation effects. The results imply the biological influences of PTM-Aß variants on the formation of amyloid deposits through cross-seeded fibrillization.

Fibrillization of 40-Residue ß-Amyloid Peptides in Membrane-Like Environments Leads to Different Fibril Structures and Reduced Molecular Polymorphisms.

The molecular-level polymorphism in ß-Amyloid (Aß) fibrils have recently been considered as a pathologically relevant factor in Alzheimer's disease (AD). Studies showed that the structural deviations in human-brain-seeded Aß fibrils potentially correlated with the clinical histories of AD patients. For the 40-residue Aß (Aß40) fibrils derived from human brain tissues, a predominant molecular structure was proposed based on solid-state nuclear magnetic resonance (ssNMR) spectroscopy. However, previous studies have shown that the molecular structures of Aß 40 fibrils were sensitive to their growth conditions in aqueous environments. We show in this work that biological membranes and their phospholipid bilayer mimics serve as environmental factors to reduce the structural heterogeneity in Aß40 fibrils. Fibrillization in the presence of membranes leads to fibril structures that are significantly different to the Aß40 fibrils grown in aqueous solutions. Fibrils grown from multiple types of membranes, including the biological membranes extracted from the rats' synaptosomes, shared similar ssNMR spectral features. Our studies emphasize the biological relevance of membranes in Aß40 fibril structures and fibrillization processes.

Effect of Post-Translational Modifications and Mutations on Amyloid-ß Fibrils Dynamics at N Terminus.

We investigate the variability in the dynamics of the disordered N-terminal domain of amyloid-ß fibrils (Aß), comprising residues 1-16 of Aß1-40, due to post-translational modifications and mutations in the ß-bend regions known to modulate aggregation properties. Using 2H static solid-state NMR approaches, we compare the dynamics in the wild-type Aß fibrils in the threefold symmetric polymorph with the fibrils from three post-translational modification sequences: isoaspartate-D7, the phosphorylation of S8, and an N-terminal truncation ΔE3. Additional comparisons are made with the mutants in the ß-bend region (residues 21-23) corresponding to the familial Osaka E22Δ deletion and D23N Iowa mutation. We also include the aggregates induced by Zn2+ ions. The dynamics are probed at the F4 and G9 positions. The main motional model involves two free states undergoing diffusion and conformational exchanges with the bound state in which the diffusion is quenched because of transient interactions involving fibril core and other intrastrand contacts. The fraction of the bound state increases in a sigmoidal fashion with a decrease in temperature. There is clear variability in the dynamics: the phosphorylation of S8 variant is the most rigid at the G9 site in line with structural studies, the ΔE3 fibrils are more flexible at the G9 site in line with the morphological fragmentation pattern, the Zn-induced aggregates are the most mobile, and the two ß-bend mutants have the strongest changes at the F4 site toward higher rigidity. Overall, the changes underlie the potential role of conformational ensembles in setting the stage for aggregation-prone states.

PLD1 promotes dendritic spine morphogenesis via activating PKD1.

Dendritic spines on the dendrites of pyramidal neurons are one of the most important components for excitatory synapses, where excitatory information exchanges and integrates. The defects of dendritic spine development have been closely connected with many nervous system diseases including autism, intellectual disability and so forth. Based on our previous studies, we here report a new functional signaling link between phospholipase D1 (PLD1) and protein kinase D1 (PKD1) in dendritic spine morphogenesis. Coimmunoprecipitation assays showed that PLD1 associates with PKD1. A series of knocking down and rescuing experiments demonstrated that PLD1 acts upstream of PKD1 in positively regulating dendritic spine morphogenesis. Using PLD1 inhibitor, we found that PLD1 activates PKD1 to promote dendritic spine morphogenesis. Thus, we further reveal the roles of the two different enzymes in neuronal development.

Molecular structure of an N-terminal phosphorylated ß-amyloid fibril.

The structural polymorphism in ß-amyloid (Aß) plaques from Alzheimer disease (AD) has been recognized as an important pathological factor. Plaques from sporadic AD patients contain fibrillar deposits of various amyloid proteins/peptides, including posttranslational modified Aß (PTM-Aß) subtypes. Although many PTM-Aßs were shown to accelerate the fibrillation process, increase neuronal cytotoxicity of aggregates, or enhance the stability of fibrils, the contribution of PTM-Aßs to structural polymorphisms and their pathological roles remains unclear. We report here the NMR-based structure for the Ser-8-phosphorylated 40-residue Aß (pS8-Aß40) fibrils, which shows significant difference to the wild-type fibrils, with higher cross-seeding efficiency and thermodynamic stability. Given these physicochemical properties, the structures originated from pS8-Aß40 fibrils may potentially dominate the polymorphisms in the mixture of wild-type and phosphorylated Aß deposits. Our results imply that Aß subtypes with "seeding-prone" properties may influence the polymorphisms of amyloid plaques through the cross-seeding process.

The on-fibrillation-pathway membrane content leakage and off-fibrillation-pathway lipid mixing induced by 40-residue ß-amyloid peptides in biologically relevant model liposomes.

Disruption of the synaptic plasma membrane (SPM) induced by the aggregation of ß-amyloid (Aß) peptides has been considered as a potential mechanism for the neurotoxicity of Aß in Alzheimer's disease (AD). However, the molecular basis of such membrane disruption process remains unclear, mainly because of the severe systematic heterogeneity problem that prevents the high-resolution studies. Our previous studies using a two-component phosphatidylcholine (PC)/phosphatidylglycerol (PG) model liposome showed the presence of Aß-induced membrane disruptions that were either on the pathway or off the pathway of fibril formation. The present study focuses on a more biologically relevant model membrane with compositions that mimic the outer leaflet of SPMs. The main findings are: (1) the two competing membrane disruption effects discovered in PC/PG liposomes and their general peptide-to-lipid-molar-ratio dependence persist in the more complicated membrane models; (2) the SPM-mimic membrane promotes the formation of certain "on-fibrillation-pathway" intermediates with higher α-helical structural population, which lead to more rapid and significant of membrane content leakage; (3) although the "on-fibrillation-pathway" intermediate structures show dependence on membrane compositions, there seems to be a common final fibril structure grown from different liposomes, suggesting that there may be a predominant fibril structure for 40-residue Aß (i.e. Aß40) peptides in biologically-relevant membranes. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.

Prophylactic Vaccine Based on Pyroglutamate-3 Amyloid ß Generates Strong Antibody Response and Rescues Cognitive Decline in Alzheimer's Disease Model Mice.

Clearance of amyloid ß (Aß) by immunotherapy is one of the fancy methods to treat Alzheimer's disease (AD). However, the failure of some clinical trials suggested that there may be something ignored in the past development of immunotherapy. Pyroglutamate-3 Aß (AßpE3-X), which was found to be abundant in the patients' brain, has attracted much attention after the report that AßpE3-42 could serve as a template to exacerbate the aggregation of Aß. In addition, AßpE3-X could not be recognized by the antibodies targeting the N-terminus of Aß, suggesting that AßpE3-X maybe the ignored one. Indeed, passive immunization targeting AßpE3-X has shown some beneficial results, while active immunotherapy has not been extensively studied. In the present study, we designed and synthesized a novel peptide vaccine targeting AßpE3-X, which contains AßpE3-15 as B cell epitope and P2 as T cell epitope. We showed that this vaccine could induce strong antibody response to AßpE3-X. We also showed that prophylactic immunization of AD model mice with our vaccine could reduce Aß plaques and rescue cognitive decline. This new kind of Aß vaccine will open up new directions for AD immunotherapy.

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 at Ser8 as an Intrinsic Regulatory Switch to Regulate the Morphologies and Structures of Alzheimer's 40-residue ß-Amyloid (Aß40) Fibrils.

Polymorphism of amyloid-ß (Aß) fibrils, implying different fibril structures, may play important pathological roles in Alzheimer's disease (AD). Morphologies of Aß fibrils were found to be sensitive to fibrillation conditions. Herein, the Ser8-phosphorylated Aß (pAß), which is assumed to specially associate with symptomatic AD, is reported to modify the morphology, biophysical properties, cellular toxicity, and structures of Aß fibrils. Under the same fibrillation conditions, pAß favors the formation of fibrils (Fpß), which are different from the wild-type Aß fibrils (Fß). Both Fß and Fpß fibrils show single predominant morphologies. Compared with Fß, Fpß exhibits higher propagation efficiency and higher neuronal cell toxicity. The residue-specific structural differences between the Fß- and Fpß-seeded Aß fibrils were identified using magic angle spin NMR. Our results suggest a potential regulatory mechanism of phosphorylation on Aß fibril formation in AD and imply that the post-translationally modified Aß, especially the phosphorylated Aß, may be an important target for the diagnosis or treatment of AD at specific stages.

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.

MiR-639 promoted cell proliferation and cell cycle in human thyroid cancer by suppressing CDKN1A expression.

Accumulating evidence has indicated that aberrantly expressed microRNAs (miRs) are extensively involved in cancer development and progression. MiR-639 has been reported to act as tumor promoter in various types of cancer. However, the biological function and underlying molecular mechanism of miR-639 in thyroid carcinoma (TC) have not been intensively investigated. Herein the present study aimed to investigate the functional role of miR-639 in TC. We found that miR-639 expression was upregulated in TC cells and clinical tissues. Overexpression of miR-639 promoted TC cell proliferation and cell cycle, with increased expression of CyclinE and c-myc, whereas miR-639-in reverses the function. Using prediction software and luciferase reporter assay, we found that CDKN1A was a target of miR-639. CDKN1A small interfering RNA (siRNA) abrogated the role of miR-639-in on cell proliferation of TC. In summary, our data demonstrated that miR-639 upregulation was associated with development of TC, miR-639 promoted cell proliferation and cell cycle by targeting CDKN1A in TC.

Clearance of the intracellular high level of the tau protein directed by an artificial synthetic hydrolase.

Promoting clearance of intracellular excessive tau is a potential therapeutic strategy for treating Alzheimer's disease. In this work, we designed and synthesized a cyclen-hybrid artificial 'hydrolase' I1-Cu(II) to cleave tau in vitro. Furthermore, a cell-permeable 'hydrolase' I2-Cu(II), derived from I1-Cu(II), was also synthesized to cleave intracellular tau proteins.

Addition of artificial salt bridge by Ile646Lys mutation in gp41 coiled-coil domain regulates 6-helical bundle formation.

HIV entry is mediated by the envelope glycoproteins gp120 and gp41. The gp41 subunit contains several functional domains: the N-terminal heptad repeat (NHR) domains fold a triple stranded coiled-coil forming a meta-stable prefusion intermediate. C-terminal heptad repeat (CHR) subsequently folds onto the hydrophobic grooves of the NHR coiled-coil to form a stable 6-helix bundle, which juxtaposes the viral and cellular membranes for fusion. The C34 which has 34 amino acid residues is known as the core structure in CHR. A highly anti-HIV peptide inhibitor derived from C34 was designed. An artificial salt bridge was added in the 6-helical bundle by substitution of lysine for Ile646. With a cholesterol modification at C-terminal, the inhibitor containing I646K mutation represented higher anti-viral activity than C34-cholesterol combination without mutation.