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One of the most successful strategies in designing peptide‐based cancer vaccines is modifying natural epitope peptides to increase their binding strength to human leukocyte antigens (HLAs). Anchor‐modified Mart‐1 peptide (ELAGIGILTV) is among the artificial epitope peptides with the highest binding affinity for HLA‐A*0201. In this study, by fluorescence labeling of its either C‐ or N‐terminus with Nε‐(5‐carboxyfluorescein)‐l‐lysine, we not only made it traceable but also drastically increased its binding strength to HLA‐A*0201. HLA streptamer, for the first time, is introduced for measuring the binding constants (Ka) of the labeled peptides. The affinity of the labeled peptides for the HLA‐A*201 of the MCF‐7 cells was extraordinarily high and co‐incubating them with the highest possible amount of the unlabeled peptide, as a competitor, did not significantly prohibit them from binding to the HLA. The reproducibility of the obtained results was confirmed by using the T2 cell line. The HLA‐deficient K562 cell line was used as the negative control. With in silico simulations, we found two hydrophobic pockets on both sides of HLA‐A*0201 for anchoring the C‐ or N‐terminal 5‐carboxyfluorescein probe, which can explain the extraordinary affinity of the labeled peptides for the HLA‐A*0201. [ABSTRACT FROM AUTHOR]

pKa values of homorepeat hexapeptides with a 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) chromophore attached at the peptide C termini, through an asparagine derivative (Dbo), namely His6–Dbo (H6), Lys6–Dbo (K6), and Arg6–Dbo (R6), were determined by a novel fluorescence-based method. The fluorescence lifetime of Dbo in the peptides (τ) was measured as a function of pH. The side chains collide with Dbo intramolecularly and quench it efficiently only when they are deprotonated (i.e., pH ≥ side chain pKa). The pKa values of the H6, K6, and R6 peptides, attributable to side chain ionization, were found to be depressed compared to the pKa values of the His, Lys, and Arg residues in their free amino acid forms. We further looked into the structural changes of the peptides by molecular dynamics (MD) simulations; the peptides were structurally more expanded when their side chains are protonated. The structural expansion of the peptides reflects an electrostatic repulsion between the protonated side chain residues, which also accounts for the observed decrease in pKa values, which corresponds to a facilitated deprotonation, assisted by electrostatic repulsion. [ABSTRACT FROM AUTHOR]

Der Makrocyclus p ‐ Sulfonatocalix[4]aren (CX4) und der Fluoreszenzfarbstoff Lucigenin (LCG) bilden einen stabilen Wirt/Gast ‐ Komplex, in dem die Fluoreszenz des Farbstoffs gelöscht wird. Die Inkubation von lebenden V79 ‐ und CHO ‐ Zellen mit dem chemosensorischen Ensemble CX4/LCG führt zu dessen spontaner Aufnahme. Die nachfolgende Zugabe von Cholin, Acetylcholin oder Protamin, die alle eine hohe Affinität für CX4 haben und in der Lage sind, in die Zellen einzudringen, führte zu einer positiven Fluoreszenzantwort der Zellen. Dies kann auf die Verdrängung des LCG aus CX4 durch die Analyten zurückgeführt werden. Die Ergebnisse zeigen die grundsätzliche Funktionsfähigkeit von Indikatorverdrängungsassays mit synthetisch hergestellten Rezeptoren zur Detektion der Aufnahme bioorganischer Analyten in lebenden Zellen. Fast wie in der Küvette können Wirt/Farbstoff ‐ Komplexe dazu verwendet werden, die Verdrängung durch einen Analyten in lebenden Zellen zu verfolgen. Dementsprechend werden p ‐ Sulfonatocalix[4]aren (CX4) und der Fluoreszenzfarbstoff Lucigenin (LCG) in CHO ‐ und V79 ‐ Zellen aufgenommen und bilden im Zytoplasma den schwach fluoreszierenden CX4/LCG ‐ Komplex. Die Zugabe von Analyten (Acetylcholin, Cholin und Protamin) führt zu einem deutlichen Fluoreszenzanstieg. [ABSTRACT FROM AUTHOR]

The macrocycle p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) form a stable host-guest complex, in which the dye fluorescence is quenched. Incubation of live V79 and CHO cells with the CX4/LCG chemosensing ensemble resulted in its spontaneous uptake. Subsequent addition of choline, acetylcholine, or protamine, which have a high affinity for CX4 and are capable of entering cells, resulted in a fluorescence switch-on response. This can be traced to the displacement of LCG from CX4 by the analytes. The results establish the principal functionality of indicator displacement assays with synthetic receptors for the detection of the uptake of bioorganic analytes by live cells. [ABSTRACT FROM AUTHOR]

Coulomb repulsion between like-chargedside chains is presentlyviewed as a major force that impacts the biological activity of intrinsicallydisordered polypeptides (IDPs) by determining their spatial dimensions.We investigated short synthetic models of IDPs, purely composed ofionizable amino acid residues and therefore expected to display anextreme structural and dynamic response to pH variation. Two synergistic,custom-made, time-resolved fluorescence methods were applied in tandemto study the structure and dynamics of the acidic and basic hexapeptidesAsp6, Glu6, Arg6, Lys6, and His6between pH 1 and 12. (i) End-to-end distanceswere obtained from the short-distance Förster resonance energytransfer (sdFRET) from N-terminal 5-fluoro-l-tryptophan (FTrp)to C-terminal Dbo. (ii) End-to-end collision rates were obtained forthe same peptides from the collision-induced fluorescence quenching(CIFQ) of Dbo by FTrp. Unexpectedly, the very high increase of chargedensity at elevated pH had no dynamical or conformational consequencein the anionic chains, neither in the absence nor in the presenceof salt, in conflict with the common view and in partial conflictwith accompanying molecular dynamics simulations. In contrast, thecationic peptides responded to ionization but with surprising patternsthat mirrored the rich individual characteristics of each side chaintype. The contrasting results had to be interpreted, by consideringsalt screening experiments, N-terminal acetylation, and simulations,in terms of an interplay of local dielectric constant and peptide-lengthdependent side chain charge–charge repulsion, side chain functionalgroup solvation, N-terminal and side chain charge–charge repulsion,and side chain–side chain as well as side chain–backboneinteractions. The common picture that emerged is that Coulomb repulsionbetween water-solvated side chains is efficiently quenched in shortpeptides as long as side chains are not in direct contact with eachother or the main chain. [ABSTRACT FROM AUTHOR]

Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed. [ABSTRACT FROM AUTHOR]

A novel cell-penetrating peptide (CPP) called FAM -Y 4 R 4 , with FAM as a fluorescent probe, was developed. Initially, we aimed to use Y 4 as a supramolecular host for water-insoluble drugs, with R 4 driving the complex into cells. However, an unexpected hurdle was discovered; the peptide self-assembled into amorphous aggregates, rendering it ineffective for our intended purpose. Molecular dynamics simulations revealed that intermolecular cation-π interactions between arginine and tyrosine caused this aggregation. By decorating the R 4 sidechains with p -sulfonatocalix[4]arene (CX4), we successfully dissolved most of the aggregates, significantly improved the peptide's solubility and enhanced the cell uptake with MCF7 and A549 cells via both direct penetration and endocytosis. The binding strength between CX4 and R 4 , as well as the interaction between curcumin and tyrosines was quantified. Encouragingly, our results showed that FAM -Y 4 R 4 , with CX4, effectively delivered curcumin - as a model for poorly water-soluble drugs - into cells which exhibited potent anticancer activity. Using R 4 /CX4 instead of the conventional R 7-9 oligoarginine-based CPP simplifies peptide synthesis and offers higher yields. CX4 shows promise for addressing aggregation issues in other peptides that undergo a similar aggregation mechanism., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Acute lymphoblastic leukemia (ALL) is a malignancy of bone marrow lymphoid precursors. Despite effective treatments, the causes of its progression or recurrence are still unknown. Finding prognostic biomarkers is needed for early diagnosis and more effective treatment. This study was performed to identify long non-coding RNAs (lncRNAs) involved in ALL progression by constructing a competitive endogenous RNA (ceRNA) network. These lncRNAs may serve as potential new biomarkers in the development of ALL. The GSE67684 dataset identified changes in lncRNAs and mRNAs involved in ALL progression. Data from this study were re-analyzed, and probes related to lncRNAs were retrieved. Targetscan, miRTarBase, and miRcode databases were used to identify microRNAs (miRNAs) related to the discovered genes and lncRNAs. The ceRNA network was constructed, and the candidate lncRNAs were selected. Finally, the results were validated with reverse transcription quantitative real-time PCR (RT-qPCR). The ceRNA network outcomes demonstrated that the top lncRNAs associated with altered mRNAs in ALL are IRF1-AS1, MCM3AP-AS1, TRAF3IP2-AS1, HOTAIRM1, CRNDE, and TUG1. Investigations of the subnets linked to MCM3AP-AS1, TRAF3IP2-AS1, and IRF1-AS1 indicated that these lncRNAs were considerably related to pathways associated with inflammation, metastasis, and proliferation. Higher expression levels of IRF1-AS1, MCM3AP-AS1, TRAF3IP2-AS1, CRNDE, and TUG1 were found in ALL samples compared to controls. The expression of MCM3AP-AS1, TRAF3IP2-AS1, and IRF1-AS1 is significantly elevated during the progression of ALL, playing an oncogenic role. Due to their role in the main cancer pathways, lncRNAs could be suitable therapeutic and diagnostic targets in ALL., Competing Interests: The authors declare that they have no conflicts of interest to report regarding the present study., (© 2022 Nekoeian et al.)

The structural and dynamic properties of a flexible peptidic chain codetermine its biological activity. These properties are imprinted in intrachain site-to-site distances as well as in diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Förster resonance energy transfer (FRET) between two chain sites labeled with a FRET donor and acceptor. Both could be obtained from time-resolved FRET measurements if their individual contributions to the FRET efficiency could be systematically varied. Because the FRET diffusion enhancement (FDE) depends on the donor-fluorescence lifetime, it has been proposed that the FDE can be reduced by shortening the donor lifetime through an external quencher. Benefiting from the high diffusion sensitivity of short-distance FRET, we tested this concept experimentally on a (Gly-Ser)(6) segment labeled with the donor/acceptor pair naphthylalanine/2,3-diazabicyclo[2.2.2]oct-2-ene (NAla/Dbo). Surprisingly, the very effective quencher potassium iodide (KI) had no effect at all on the average donor-acceptor distance, although the donor lifetime was shortened from ca. 36 ns in the absence of KI to ca. 3 ns in the presence of 30 mM KI. We show that the proposed approach had to fail because it is not the experimentally observed but the radiative donor lifetime that controls the FDE. Because of that, any FRET ensemble measurement can easily underestimate diffusion and might be misleading even if it employs the Haas-Steinberg diffusion equation (HSE). An extension of traditional FRET analysis allowed us to evaluate HSE simulations and to corroborate as well as generalize the experimental results. We demonstrate that diffusion-enhanced FRET depends on the radiative donor lifetime as it depends on the diffusion coefficient, a useful symmetry that can directly be applied to distinguish dynamic and structural effects of viscous cosolvents on the polymer chain. We demonstrate that the effective FRET rate and the recovered donor-acceptor distance depend on the quantum yield, most strongly in the absence of diffusion, which has to be accounted for in the interpretation of distance trends monitored by FRET.