Polypeptide Sequence Research Articles

Single nucleotide translation without ribosomes.

The translation of messenger RNA sequences into polypeptide sequences according to the genetic code is central to life. How this process, which relies on the ribosomal machinery, arose from much simpler precursors is unclear. Here, we demonstrate that single nucleotides charged with an amino acid couple with amino acids linked to the 5'-terminus of an RNA primer in reactions directed by the nucleotides of an RNA template in dilute aqueous solution at 0 °C. When a mixture of U-Val, A-Gly and G-Leu competed for coupling to Gly-RNA, base pairing dictated which dipeptide sequence formed preferentially. The resulting doubly anchored dipeptides can retain their link to the primer for further extension or can be fully released under mild acidic conditions. These results show that a single-nucleotide-based form of translation exists that requires no more than oligoribonucleotides and anchored amino acids.

Constructing Brain Aβ-Targeting Nanoparticles Loaded with EGCG for Treating Alzheimer's Disease in Mice

To construct a nanodelivery system surface-modified with RD2 peptide (polypeptide sequence PTLHTHNRRRRR) for brain tissue penetration and β-amyloid (Aβ) binding. Epigallocatechin-3-gallate (EGCG) was selected for encapsulation in the targeted delivery system and its therapeutic potential for Alzheimer's disease (AD) was investigated. EGCG-load nanoparticles (NP/EGCG), NP/EGCG with RD2 peptide surface modification (RD2-NP/EGCG), as well as RD2 peptide-modified blank nanoparticles (RD2-NP) were prepared and characterized. Thioflavin T assay was done to assess the ability of RD2-NP to bind with Aβ and ex vivo imaging was conducted to evaluate the distribution of RD2-NP in brain lesion sites. The AD mice model was established by injecting oligomeric Aβ 42 in the bilateral hippocampi of ICR mice. Then AD mice were administered intravenously through the tail vein with normal saline, EGCG solution, NP/EGCG or RD2-NP/EGCG for 28 d, respectively, and the Morris water maze tests were performed to assess the spatial memory of mice. Subsequently, RT-PCR method was used to determine the mRNA levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the hippocampus of the mice, and the morphological changes of hippocampal neurons were observed with Nissl staining. Additionally, the pathological changes of heart, liver, spleen, lung, and kidney were characterized by hematoxylin-eosin (HE) staining. The particle diameter of the prepared RD2-NP/EGCG was (204.83±2.80) nm and the zeta potential was -23.88 mV. The encapsulation efficiency and drug loading capacity were 94.39% and 5.90%, respectively. The RD2 peptide modification has no significant effect on the physiochemical properties of the nanoparticles. RD2-NP had good Aβ binding ability, and it could be concentrated in hippocampus and cerebral cortex, the most common Aβ deposition sites. The four-week RD2-NP/EGCG treatment significantly decreased the expression of the pro-inflammatory cytokine TNF-α and IL-1β, restored neuronal losses and hippocampal damage, and ameliorated spatial memory impairment in AD model mice. Moreover, treatment with the RD2-NP/EGCG did not present organ toxicity. Surface modified RD2 peptide nanodelivery system can efficiently deliver drugs to AD lesions and improve the therapeutic effect of EGCG on AD.

Bacterial Protein Homeostasis Disruption as a Therapeutic Intervention.

Cells have evolved a complex molecular network, collectively called the protein homeostasis (proteostasis) network, to produce and maintain proteins in the appropriate conformation, concentration and subcellular localization. Loss of proteostasis leads to a reduction in cell viability, which occurs to some degree during healthy ageing, but is also the root cause of a group of diverse human pathologies. The accumulation of proteins in aberrant conformations and their aggregation into specific beta-rich assemblies are particularly detrimental to cell viability and challenging to the protein homeostasis network. This is especially true for bacteria; it can be argued that the need to adapt to their changing environments and their high protein turnover rates render bacteria particularly vulnerable to the disruption of protein homeostasis in general, as well as protein misfolding and aggregation. Targeting bacterial proteostasis could therefore be an attractive strategy for the development of novel antibacterial therapeutics. This review highlights advances with an antibacterial strategy that is based on deliberately inducing aggregation of target proteins in bacterial cells aiming to induce a lethal collapse of protein homeostasis. The approach exploits the intrinsic aggregation propensity of regions residing in the hydrophobic core regions of the polypeptide sequence of proteins, which are genetically conserved because of their essential role in protein folding and stability. Moreover, the molecules were designed to target multiple proteins, to slow down the build-up of resistance. Although more research is required, results thus far allow the hope that this strategy may one day contribute to the arsenal to combat multidrug-resistant bacterial infections.

Dissection of phospholipases A2 reveals multifaceted peptides targeting cancer cells, Leishmania and bacteria

Cationic peptides bio-inspired by natural toxins have been recognized as an efficient strategy for the treatment of different health problems. Due to the specific interaction with substrates from biological membranes, snake venom phospholipases (PLA2s) represent valuable scaffolds for the research and development of short peptides targeting parasites, bacteria, and cancer cells. Considering this, we evaluated the in vitro therapeutic potential of three biomimetic peptides (pCergo, pBmTxJ and pBmje) based on three different amino acid sequences from Asp49 PLA2s. First, short amino acid sequences (12–17 in length) derived from these membranolytic toxins were selected using a combination of bioinformatics tools, including AntiCP, AMPA, PepDraw, ToxinPred, and HemoPI. The peptide, from each polypeptide sequence, with the greatest average antimicrobial index, no toxicity, and no hemolysis predicted was synthesized, purified, and characterized. According to in vitro assays performed, pBmje showed moderate cytotoxicity specifically against MCF–7 (breast cancer cells) with an EC50 of 464.85 µM, whereas pBmTxJ showed an antimicrobial effect against Staphylococcus aureus (ATCC 25923) with an MIC of 37.5 µM, and pCergo against E. coli (ATCC 25922) with an MIC of 75 µM. In addition, pCergo showed antileishmanial activity with an EC50 of 93.69 µM and 110.40 µM against promastigotes of Leishmania braziliensis and L. amazonensis, respectively. Altogether, these results confirmed the versatility of PLA2-derived synthetic peptides, highlighting the relevance of the use of these membrane-interacting toxins as specific archetypes for drug design focused on public health problems.

Insights into the miscibility characteristics of plastic-mimetic polypeptide with hydroxypropylmethylcellulose: Investigation of thermal degradability and intermolecular interactions

In this investigation, we integrated the parent recurring sequence of the plastic-derived polypeptide, poly[0.8(AVGVP),0.2(AEGVP)] (A, V, G, P, and E represents Alanine, Valine, Glycine, Proline, and Glutamic acid respectively) followed by characterization with inverse transition temperature, 13C, and 1H-NMR spectroscopy. The miscibility attributes of poly[0.8(AVGVP),0.2(AEGVP)] with Hydroxypropylmethylcellulose was examined both in aqueous and solid-phase. The Huggins’ co-efficient [KH], the intrinsic viscosity [η], the interaction parameters ΔB and μ suggested by Chee, ΔK and β recommended by Jiang and Han, α by Sun, Δ[η]m by Garcia showed that the polypeptide was miscible with HPMC in all proportions. DSC studies revealed single Tg values, and TGA manifested the enhanced thermal stability for all the proportions compared with their individuals. Further, verified the results by SEM and XRD. The FTIR evidenced existence of intermolecular hydrogen bonding between the two constituent polymers that caused the miscible blend system.

Biomolecular Condensates under Extreme Martian Salt Conditions

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming nonmembranous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the subseafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.

ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry.

Top-down mass spectrometry (TD-MS) of peptides and proteins results in product ions that can be correlated to polypeptide sequence. Fragments can either be terminal fragments, which contain either the N- or the C-terminus, or internal fragments that contain neither termini. Normally, only terminal fragments are assigned due to the computational difficulties of assigning internal fragments. Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complementary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum. Data are available via the MassIVE community resource with the identifiers MSV000086788 and MSV000086789.

A distinct molecular signature on anhydrobiotic cyanobacterial metallothioneins

Anhydrobiosis refers to a state of suspended animation in which some organisms enter when exposed to extreme desiccation, ensuring them an outstanding tolerance to several physical stresses due to molecular and cellular adaptations. Metallothioneins (MTs) are short cysteine-rich metal-chelating proteins that work as a cellular protection element in metal ion-rich conditions. Here we aimed to investigate possible molecular signatures in primary and tertiary structures in anhydrobiotic cyanobacterial MTs. Anhydrobiotic and non-anhydrobiotic cyanobacterial MT amino acid sequences were retrieved from NCBI database and aligned in Clustal Omega server. Additionally, the amino acid compositions of these sequences were determined by GeneRunner. Further, we carried out homology-modeling via SWISS-MODEL, structural superposition in UCSF Chimera 1.4 Matchmaker tool and ligand-binding site prediction via COFACTOR. In silico analyses revealed specific divergences in amino acid positions between MT groups, evidencing positive and negative selections, however without affecting final protein structures. Some of these changes on polypeptide sequence potentially enhance protein stabilization during desiccation, whereas others possibly act as additional metal-ion coordinating residues. Analyses on the molecular adaptations on anhydrobiotic cyanobacterial MTs help shed light on their molecular functions and biological roles, as well as may have applications on the development of desiccation- and metal-tolerant organisms.

Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3

Amino acid substitutions within the conserved polypeptide sequence of the insect olfactory receptor co-receptor (Orco) have been demonstrated to influence its pharmacological properties. By sequence analysis and phylogenetic investigation, in the Lepidopteran subgroup Ditrysia we identified a fixed substitution in the intracellular loop-3 (ICL-3) of a conserved histidine to glutamine. By means of HEK293 cells as a heterologous system, we functionally expressed Orco from the Ditrysian model Cydia pomonella (CpomOrco) and compared its functional properties with a site-directed mutagenized version where this ICL-3-glutamine was reverted to histidine (CpomOrcoQ417H). The mutagenized CpomOrcoQ417H displayed decreased responsiveness to VUAA1 and reduced response efficacy to an odorant agonist was observed, when co-transfected with the respective OR subunit. Evidence of reduced responsiveness and sensitivity to ligands for the mutagenized Orco suggest the fixed glutamine substitution to be optimized for functionality of the cation channel within Ditrysia. In addition, contrary to the wild type, the mutagenized CpomOrcoQ417H preserved characteristics of VUAA-binding when physiologic conditions turned to acidic. Taken together, our findings provide further evidence of the importance of ICL-3 in forming basic functional properties of insect Orco- and Orco/OR-channels, and suggest involvement of ICL-3 in the potential functional adaptation of Ditrysian Orcos to acidified extra-/intracellular environment.

PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high-throughput sequencing.

The reconstructed in vitro translation system known as the PURE system has been used in a variety of cell-free experiments such as the expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE-based display method for the preparation of stable messenger RNA (mRNA) and complementary DNA (cDNA)-peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry-over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. We chose the commercially available anti-FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 × 1012 random sequences, a round-by-round high-throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG epitope motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr (Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, the comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. We also show that gel purification steps in the refined PURE-based display method improve conjugate formation efficiency and enhance the enrichment rate of FLAG epitope motifs in later rounds of selection especially for mRNA display. Overall, the generalized procedure and consistent performance of two different display methods achieved by the commercially available PURE system will be useful for future studies to explore the sequence and functional space of diverse polypeptides.

Functional Roles of the von Willebrand Factor Propeptide.

The primary polypeptide sequence of von Willebrand factor (VWF) includes an N-terminal 741-amino acid VWF propeptide (VWFpp). In cells expressing VWF, the VWFpp performs two critical functions. In the Golgi, VWFpp mediates the intermolecular disulfide linkages that generate high-molecular-weight VWF multimers. Subsequently, the VWFpp, which is proteolytically cleaved from mature VWF by furin, functions to generate the endothelial storage organelles (Weibel-Palade bodies) in which VWF and a distinct collection of proteins are stored, and from where they undergo regulated secretion from the endothelium. The VWFpp is secreted from endothelial cells as dimers and circulates in plasma with at least some of the dimers associating with a noncovalent manner with the D'D3 domain of mature VWF. The VWFpp has a half-life of 2 to 3 hours in plasma, but to date no extracellular function has been determined for the molecule. Nevertheless, its large size and several biologically interesting structural features (two sets of vicinal cysteines and an RGD sequence) suggest that there may be roles that the VWFpp plays in hemostasis or associated physiological processes such as angiogenesis or wound repair.

Modelling the effect of ribosome mobility on the rate of protein synthesis

Translation is one of the main steps in the synthesis of proteins. It consists of ribosomes that translate sequences of nucleotides encoded on mRNA into polypeptide sequences of amino acids. Ribosomes bound to mRNA move unidirectionally, while unbound ribosomes diffuse in the cytoplasm. It has been hypothesized that finite diffusion of ribosomes plays an important role in ribosome recycling and that mRNA circularization enhances the efficiency of translation, see e.g. Lodish et al. (Molecular cell biology, 8th edn, W.H. Freeman and Company, San Francisco, 2016). In order to estimate the effect of cytoplasmic diffusion on the rate of translation, we consider a totally asymmetric simple exclusion process coupled to a finite diffusive reservoir, which we call the ribosome transport model with diffusion. In this model, we derive an analytical expression for the rate of protein synthesis as a function of the diffusion constant of ribosomes, which is corroborated with results from continuous-time Monte Carlo simulations. Using a wide range of biological relevant parameters, we conclude that diffusion is not a rate limiting factor in translation initiation because diffusion is fast enough in biological cells.Graphic abstract

Deciphering Multiphasic Structure of Protein-RNA Condensates

Intracellular biomolecular condensates, such as the nucleolus and stress granules, are phase separated fluids which commonly display complex internal sub-structures. These sub-structures are understood as a consequence of internal de-mixing of condensate components into distinct co-existing phases. Although some in vitro model systems have been successful in recapitulating such multi-phasic structures in phase-separated condensates, it remains unknown how spatial arrangement of multiple co-existing liquid phases is regulated. We answer this question by employing a three-component system, comprising of a prion-like polypeptide (PLP), an arginine-rich polypeptide (RLP), and RNA, exhibiting overlapping inter-molecular interactions. Using experiments and simulations, we show that PLP-RLP-RNA ternary system forms stable coexisting PLP and RLP-RNA condensates. We find that the topology of these bi-phasic condensates sensitively depends on the hierarchy of inter-molecular interactions at the liquid-liquid interface, which can be tuned via mixture composition and polypeptide sequence perturbations. This finding enabled us to predictively control multi-phasic morphologies, allowing us to design totally-engulfed, partially-engulfed, completely-detached, and Janus-like condensates in the PLP-RLP-RNA ternary mixture. The direct correspondence between the microscopic intermolecular interactions and the mesoscopic multiphase structuring is a manifestation of tunable interfacial tensions in the system. Our finding allows us to reliably control the multiphasic structuring, highlighting hitherto unknown physical principles that regulate the spatial organization of multiphasic biomolecular condensates.

Reviewing the Latest Findings on the Polypeptide Sequence of the SARS-CoV-2 S-Protein to Raise Questions about the Origins of RaTG13

The ongoing coronavirus disease outbreak started from a local seafood market of the Wuhan city (China). The first reported infections were six seafood street-sellers with severe pneumonia admitted to the intensive care unit of the Wuhan Jin-Yin-Tan Hospital. Upon metagenomics sequencing and phylogenetic analysis of the complete genomes of the virus isolates from these patients, RaTG13, a bat coronavirus previously detected in Rhinolophus affinis from the Chinese province of Yunnan, was identified as the most identical and closely-related coronavirus to the human pathogen, which was designated as SARS-CoV-2. The most updated evidences suggest adaptation of the human coronavirus SARS-CoV-2 from an animal reservoir, which supposedly underwent some sort of recombination. However, the processes to describe how adaptation of the virus from an animal to a human host may occur still cannot be explained. From a polypeptide sequence analysis of the S-proteins of SARS-coronaviruses, this contribution address questions about the origins of RaTG13, which still remains the most identical strain with SARS-CoV-2 described up to now.

Spatial Covariant Management of Thermodynamics Contributes to Protein Fold Function Through Quality Assurance

Abstract Although the impact of genome variation on the thermodynamic properties of the protein fold has been studied in vitro, it remains a challenge to assign these relationships across the entire polypeptide sequence in vivo. Using the Gaussian process regression-based principle of Spatial CoVariance (SCV), we globally assign on a residue-by-residue the biological thermodynamic properties contributing to the functional fold in the cell using CFTR as an example. We demonstrate the existence of a thermodynamically sensitive region of the CFTR fold involving the interface between NBD1 and ICL4 that contributes to the endoplasmic reticulum (ER) export. At the cell surface a new set of residues contribute uniquely to the management of channel function. These results support a general 'quality assurance' (QA) view of global protein fold management as an SCV principle describing the differential pre- and post-ER residue interactions contributing to compartmentalization of the energetics of the protein fold for function.

Visualizing the superfamily of metallo-β-lactamases through sequence similarity network neighborhood connectivity analysis

Protein sequence similarity networks (SSNs) constitute a convenient approach to analyze large polypeptide sequence datasets, and have been successfully applied to study a number of protein families over the past decade. SSN analysis is herein combined with traditional cladistic and phenetic phylogenetic analysis (respectively based on multiple sequence alignments and all-against-all three-dimensional protein structure comparisons) in order to assist the ancestral reconstruction and integrative revision of the superfamily of metallo-β-lactamases (MBLs). It is shown that only 198 out of 15,292 representative nodes contain at least one experimentally obtained protein structure in the Protein Data Bank or a manually annotated SwissProt entry, that is to say, only 1.3 % of the superfamily has been functionally and/or structurally characterized. Besides, neighborhood connectivity coloring, which measures local network interconnectivity, is introduced for detection of protein families within SSN clusters. This approach provides a clear picture of how many families remain unexplored in the superfamily, while most MBL research is heavily biased towards a few families. Further research is suggested in order to determine the SSN topological properties, which will be instrumental for the improvement of automated sequence annotation methods.

(Invited) Engineering Polypeptides for Controlled Assembly of Ionomer Thin Films

Increasing evidence suggests that control of ionomer thin film structures on metal surfaces is pivotal for efficient performance in electrochemical devices such as fuel cells and electrolyzers. Unfortunately, the assembly of these thin film structures is difficult to control using conventional methods and ideal arrangements remain unknown. Engineered polypeptides have emerged as powerful biomolecular tools in electrode assembly because binding sites and polypeptide structures can be easily modulated by changing the amino acid sequence. However, no studies have been conducted showing polypeptides can be engineered to interact with ionomers, attach them to metal surfaces, and control their arrangement. Our lab has recently developed this technology, using an elastin-like polypeptide to bind to metals, and bind to acidic and basic ionomer via ionic interactions. We use a quartz crystal microbalance with dissipation to provide detailed information about the loading, thickness, and binding behavior of the polypeptide and ionomer layers. We also use atomic force microscopy and grazing-incidence small-angle X-ray scattering to understand the impact of the biomolecules on ionomer phase separation. Finally, we have begun to analyze the performance (ionic conductivity) of the assembled films using interdigitated electrodes and electrochemical impedance spectroscopy. Through these techniques, we show that 1) our biomolecular system is highly flexible, easily adapting to different materials, 2) the polypeptide sequence can dictate ionomer phase separated structures, and 3) this system can be used to improve the performance of ionomer thin films and gain structure-function understanding. Generally, our results demonstrate that engineered polypeptides are promising tools for ionomer control in electrode engineering.

A Protease-Mediated Regulatory Chimeric Antigen Receptor (CAR): “Double Arm” CAR System Improves Tumor-Cell-Specificity of CAR-T Cell Therapy

【 Introduction 】 CAR-T cell therapy has shown excellent therapeutic effects against some malignancies including refractory B cell lymphoma or leukemia. This adoptive T cell transfer therapy is an attractive methodology; however, there are several disadvantages to be overcome. Current CAR-T cell therapy targets single cell-surface molecule, which leads to damage of normal cells expressing the target protein. Such "On-target / off-tumor" effect is one of the major adverse effects. Improvement of target-cell-specificity is needed to avoid this serious adverse effect and to expand target diseases of this therapy. Here, we show the protease-mediated "Double-Arm" CAR-T cell system, which improved the specificity of CAR-T cell therapy by recognizing two distinct cell-surface proteins. We designed two types of CARs: "Effector CAR" and "Scissors CAR". The "Effector CAR" is constituted of a single chain Fv fragment (scFv) targeting a cell-surface protein (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition polypeptide sequence, and a functional domain of CD3-zeta. The "scissors CAR" is constituted of a recognition portion targeting another protein (protein Y) and HIVPR. The HIVPR induces cleavage of the recognition polypeptide sequence in the effector CAR leading to inactivation of the effector CAR when the CAR-T cells contact with cells expressing both proteins X and Y. 【 Material and Methods 】 For proof of principle, we first constructed "anti-CD19 mCherry CAR" harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition polypeptide sequence. Also, we constructed "anti-CD19 scissors CAR", low affinity "anti-HER2 (4D5-3) scissors CAR" and high affinity "anti-HER2 (4D5-8) scissors CAR". To analyze the target-cell-dependent cleavage of mCherry CAR, 293T cells expressing these CARs were co-cultured with target cells, including K562 (CD19-, HER2-), Raji (CD19+, HER2+), or SK-BR-3 (CD19-, HER2+). To obtain target cells expressing both CD19 and HER2, Raji and SK-BR-3 cells were molecularly manipulated. (1) To evaluate efficiency of this system, after co-cultivation of CAR-transduced 293T and the target cells (K562, Raji, SK-BR-3), the localization of mCherry was examined under the microscopy and Western blotting. (2) To assess the T cell activation, we constructed "anti-CD19 effector CAR" and established Jurkat cells expressing both the "effector CAR" and the "anti-HER2 scissors CAR". These cells were co-cultured with wild type or engineered Raji or SK-BR-3 cells. T cell activation was analyzed with flowcytometric analysis. (3) To assess the CAR activity of this system, we transduced these CARs to primary T cells from healthy donors. After co-cultivation with the target cells, we measured target-specific cytotoxic activity. 【 Results 】 (1) Transduced "anti-CD19 mCherry CAR" was detected as a membrane-bound protein in 293T cells. Co-cultivation of 293T cells expressing both the "anti-CD19 mCherry CAR" and "anti-HER2 scissors CAR" with engineered Raji cells expressing both CD19 and HER2 induced cleavage of the recognition site and translocation of the mCherry from the membrane to the cytoplasm. These results suggested that this system would regulate activities of CAR-T cell through HIVPR-mediated cleavage of the "effector CAR" in vitro. (2) Jurkat cells expressing "anti-CD19 effector CAR" were activated through a target-cell-dependent manner. Furthermore, "anti-HER2 scissors CAR" attenuated T cell activation driven by "anti-CD19 effector CAR" when Jurkat cells expressing both the "anti-CD19 effector CAR" and the "anti-HER2 scissors CAR" contacted with target-cells expressing both CD19 and HER2. In addition, high affinity 4D5-8 scissors CAR showed more potent attenuation than the low affinity 4D5-3 scissors CAR. (3) Primary human T cells expressing "anti-CD19 effector CAR" showed CD19-expressing target-cell-specific cytotoxic activity. We also demonstrated that "Double Arm" primary human CAR-T cells showed tumor-cell-specificity as seen in cell-line models described above. 【Discussion】 Our "Double-Arm" CAR-T cell system has improved tumor-cell-specificity even in primary human T-cells as we expected. This system would attenuate the adverse effects of clinical CAR-T cell therapies. Disclosures No relevant conflicts of interest to declare.

Rod-shaped polypeptide nanoparticles for siRNA delivery

Rod-shaped nanoparticles have been reported to exhibit improved cellular uptake, intracellular processing and transport through tissues and organs, as compared to spherical nanoparticles. We use C-S-B triblock polypeptides composed of a collagen-like block (C), a silk-like block (S) and an oligolysine domain (B) for one-dimensional co-assembly with siRNA into rod-shaped nanoparticles. Here we investigate these siRNA encapsulating rod-shaped nanoparticles as a gene delivery system. Uptake experiments for C-S-B and C-S-B/siPlk1 particles indicate that these rod-shaped nanoparticles can efficiently deliver siPlk1 into HeLa cells. Moreover, C-S-B/siPlk1 complexes display significant mPlk1 gene knockdown in a dose-dependent manner, causing apoptosis as intended. The lower effectiveness of C-S-B/siPlk1 in inducing cell death as compared to cationic lipid-based formulations is explained by the high lysosome-C-S-B/siPlk1 co-localization ratio, which will need to be addressed in a future redesign of polypeptide sequence. Overall, the non-toxic and unique rod-shaped C-S-B nanoparticles deserve further optimization as a new siRNA delivery system for cancer therapy.

The Impact of Halogenated Phenylalanine Derivatives on NFGAIL Amyloid Formation.

The hexapeptide hIAPP22–27 (NFGAIL) is known as a crucial amyloid core sequence of the human islet amyloid polypeptide (hIAPP) whose aggregates can be used to better understand the wild‐type hIAPP′s toxicity to β‐cell death. In amyloid research, the role of hydrophobic and aromatic‐aromatic interactions as potential driving forces during the aggregation process is controversially discussed not only in case of NFGAIL, but also for amyloidogenic peptides in general. We have used halogenation of the aromatic residue as a strategy to modulate hydrophobic and aromatic‐aromatic interactions and prepared a library of NFGAIL variants containing fluorinated and iodinated phenylalanine analogues. We used thioflavin T staining, transmission electron microscopy (TEM) and small‐angle X‐ray scattering (SAXS) to study the impact of side‐chain halogenation on NFGAIL amyloid formation kinetics. Our data revealed a synergy between aggregation behavior and hydrophobicity of the phenylalanine residue. This study introduces systematic fluorination as a toolbox to further investigate the nature of the amyloid self‐assembly process.