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1.
Thermodynamics and Reaction Mechanisms for Decomposition of a Simple Protonated Tripeptide, H+GGA: From H+GGG to H+GAG to H+GGA.
Mookherjee, A, Armentrout, PB
Journal of the American Society for Mass Spectrometry. 2022;(2):355-368
Abstract
We present a thorough characterization of fragmentations observed in threshold collision-induced dissociation (TCID) experiments of protonated glycylglycylalanine (H+GGA) with Xe using a guided ion beam tandem mass spectrometer. Kinetic energy dependent cross sections for nine ionic products were obtained and analyzed to provide 0 K barriers for the five primary products, [b2]+, [y1 + 2H]+, [b3]+, [y2 + 2H]+, and [a1]+; and four secondary products, [a2]+, [a3]+, high-energy [y1 + 2H]+, and CH3CHNH2+, after accounting for multiple ion-molecule collisions, the internal energy of reactant ions, unimolecular decay rates, competition between channels, and sequential dissociations. Relaxed potential energy surface scans performed at the B3LYP-GD3BJ/6-311+G(d,p) level of theory are used to identify transition states (TSs) and intermediates of the five primary and three secondary products (with the mechanism of the other secondary product previously established). Geometry optimizations and single point energy calculations of reactants, products, intermediates, and TSs were performed at several levels of theory. These theoretical energies are compared with experimental threshold energies and found to give reasonable agreement, with B3LYP-GD3BJ and M06-2X levels of theory performing slightly better than MP2 and better than B3LYP. The results obtained here are compared with previous results for decomposition of H+GGG and H+GAG to probe the effect of changing the amino acid sequence. Methylation in H+GGA has a significant effect on the competition between the primary sequence products, [b2]+ and [y1 + 2H]+, suppressing the [b2]+ cross section by raising its threshold energy, while enhancing that of [y1 + 2H]+ by lowering its threshold energy.
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Dual intra- and extracellular release of monomethyl auristatin E from a neutrophil elastase-sensitive antibody-drug conjugate.
Mohamed Amar, IA, Huvelle, S, Douez, E, Letast, S, Henrion, S, Viaud-Massuard, MC, Aubrey, N, Allard-Vannier, E, Joubert, N, Denevault-Sabourin, C
European journal of medicinal chemistry. 2022;:114063
Abstract
Antibody-drug conjugates (ADCs) are targeted therapies, mainly used in oncology, consisting in a three-component molecular architecture combining a highly potent drug conjugated via a linker onto a monoclonal antibody (mAb), designed for the selective delivery of the drug to the tumor site. The linker is a key component, defining the ADC stability and mechanism of action, and particularly the drug release strategy. In this study, we have developed and synthesized a cleavable linker, which possesses an Asn-Pro-Val (NPV) sequence sensitive to the human neutrophil elastase (HNE), overexpressed in the tumor microenvironment. This linker permitted the site-specific conjugation of the cell-permeable drug, monomethyl auristatin E (MMAE), onto trastuzumab, using a disulfide re-bridging technology. The resulting ADC was then evaluated in vitro. This conjugate demonstrated retained antigen (Ag) binding affinity and exhibited high subnanomolar potency against Ag-positive tumor cells after internalization, suggesting an intracellular mechanism of linker cleavage. While no internalization and cytotoxic activity of this ADC was observed on Ag-negative cells in classical conditions, the supplementation of exogenous HNE permitted to restore a nanomolar activity on these cells, suggesting an extracellular mechanism of drug release in these conditions. This in vitro proof of concept tends to prove that the NPV sequence could allow a dual intra- and extracellular mechanism of drug release. This work represents a first step in the design of original ADCs with a new dual intra- and extracellular drug delivery system and opens the way to further experimentations to evaluate their full potential in vivo.
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Imaging of femtosecond bond breaking and charge dynamics in ultracharged peptides.
Eliah Dawod, I, Tîmneanu, N, Mancuso, AP, Caleman, C, Grånäs, O
Physical chemistry chemical physics : PCCP. 2022;(3):1532-1543
Abstract
X-ray free-electrons lasers have revolutionized the method of imaging biological macromolecules such as proteins, viruses and cells by opening the door to structural determination of both single particles and crystals at room temperature. By utilizing high intensity X-ray pulses on femtosecond timescales, the effects of radiation damage can be reduced. Achieving high resolution structures will likely require knowledge of how radiation damage affects the structure on an atomic scale, since the experimentally obtained electron densities will be reconstructed in the presence of radiation damage. Detailed understanding of the expected damage scenarios provides further information, in addition to guiding possible corrections that may need to be made to obtain a damage free reconstruction. In this work, we have quantified the effects of ionizing photon-matter interactions using first principles molecular dynamics. We utilize density functional theory to calculate bond breaking and charge dynamics in three ultracharged molecules and two different structural conformations that are important to the structural integrity of biological macromolecules, comparing to our previous studies on amino acids. The effects of the ultracharged states and subsequent bond breaking in real space are studied in reciprocal space using coherent diffractive imaging of an ensemble of aligned biomolecules in the gas phase.
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Interaction of KRSR Peptide with Titanium Dioxide Anatase (100) Surface: A Molecular Dynamics Simulation Study.
Tarjányi, T, Bogár, F, Minarovits, J, Gajdács, M, Tóth, Z
International journal of molecular sciences. 2021;(24)
Abstract
Due to its tensile strength and excellent biocompatibility, titanium (Ti) is commonly used as an implant material in medicine and dentistry. The success of dental implants depends on the formation of a contact between the oxidized surface of Ti implant and the surrounding bone tissue. The adsorption of proteins and peptides to the implant surface allows the bone-forming osteoblast cells to adhere to such modified surfaces. Recently, it has been observed that tetrapeptide KRSR (Lys-Arg-Ser-Arg) functionalization could promote osteoblast adhesion to implant surfaces. This may facilitate the establishment of an efficient bone-to implant contact and improve implant stability during the healing process. GROMACS, a molecular dynamics software package was used to perform a 200 ns simulation of adsorption of the KRSR peptide to the TiO2 (anatase) surface in an aqueous environment. The molecule conformations were mapped with Replica Exchange Molecular Dynamics (REMD) simulations to assess the possible peptide conformations on the anatase surface, and the umbrella sampling method was used to calculate the binding energy of the most common conformation. The simulations have shown that the KRSR peptide migrates and attaches to the surface in a stable position. The dominant amino acid residue interacting with the TiO2 surface was the N-terminal charged lysine (K) residue. REMD indicated that there is a distinct conformation that is taken by the KRSR peptide. In this conformation the surface interacts only with the lysine residue while the ser (S) and arg (R) residues interact with water molecules farther from the surface. The binding free energy of the most common conformation of KRSR peptide to the anatase (100) surface was ΔG = -8.817 kcal/mol. Our result suggests that the N-terminal lysine residue plays an important role in the adhesion of KRSR to the TiO2 surface and may influence the osseointegration of dental implants.
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β-Lactolin Enhances Neural Activity, Indicated by Event-Related P300 Amplitude, in Healthy Adults: A Randomized Controlled Trial.
Kanatome, A, Ano, Y, Shinagawa, K, Ide, Y, Shibata, M, Umeda, S
Journal of Alzheimer's disease : JAD. 2021;(2):787-796
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Abstract
BACKGROUND Epidemiological studies have shown that dairy product consumption is beneficial for cognitive function in elderly individuals. β-lactolin is a Gly-Thr-Trp-Tyr lacto-tetrapeptide rich in fermented dairy products that improves memory retrieval, attention, and executive function in older adults with subjective cognitive decline and prevents the pathology of Alzheimer's disease in rodents. There has been no study on the effects of β-lactolin on neural activity in humans. OBJECTIVE We investigated the effects of β-lactolin on neural activity and cognitive function in healthy adults. METHODS In this randomized, double-blind, placebo-controlled study, 30 participants (45-64 years old) consumed β-lactolin or placebo for 6 weeks. Neural activity during auditory and language tasks was measured through 64-channel electroencephalography. Moreover, verbal fluency tests were performed at baseline and after 6 weeks. RESULTS The β-lactolin group had a significantly higher P300 amplitude at the Cp2 site (a part of the parietal lobe near the center of brain, p = 0.011), and C4 site (the area between the frontal and parietal lobe, p = 0.02) during the auditory tasks after 6 weeks than the placebo group. Thus, β-lactolin supplementation promoted neural activity in the parietal area, which increases concentration and attention during auditory cognitive tasks. Compared with the placebo group, the β-lactolin group also showed significant changes in the scores of verbal fluency test after 6 weeks (p = 0.033). CONCLUSION Our findings provide insight into the mechanisms underlying the effects of β-lactolin on attention in healthy adults.
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The Smac mimetic BV6 cooperates with STING to induce necroptosis in apoptosis-resistant pancreatic carcinoma cells.
Hannes, S, Karlowitz, R, van Wijk, SJL
Cell death & disease. 2021;(9):816
Abstract
Pancreatic cancer (PC) still remains a major cause of cancer-related death worldwide and alternative treatments are urgently required. A common problem of PC is the development of resistance against apoptosis that limits therapeutic success. Here we demonstrate that the prototypical Smac mimetic BV6 cooperates with the stimulator of interferon (IFN) genes (STING) ligand 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP) to trigger necroptosis in apoptosis-deficient PC cells. Pharmacological inhibition of key components of necroptosis signaling, such as receptor-interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), significantly rescues PC cells from 2'3'-cGAMP/BV6/zVAD.fmk-mediated cell death, suggesting the induction of necroptosis. Consistently, 2'3'-cGAMP/BV6 co-treatment promotes phosphorylation of MLKL. Furthermore, we show that 2'3'-cGAMP stimulates the production of type I IFNs, which cooperate with BV6 to trigger necroptosis in apoptosis-deficient settings. STING silencing via siRNA or CRISPR/Cas9-mediated gene knockout protects PC cells from 2'3'-cGAMP/BV6/zVAD.fmk-mediated cell death. Interestingly, we demonstrate that nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNFα), and IFN-regulatory factor 1 (IRF1) signaling are involved in triggering 2'3'-cGAMP/BV6/zVAD.fmk-induced necroptosis. In conclusion, we show that activated STING and BV6 act together to exert antitumor effects on PC cells with important implications for the design of new PC treatment concepts.
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Inhibition of the ʟ-glutamine transporter ASCT2 sensitizes plasma cell myeloma cells to proteasome inhibitors.
Prelowska, MK, Mehlich, D, Ugurlu, MT, Kedzierska, H, Cwiek, A, Kosnik, A, Kaminska, K, Marusiak, AA, Nowis, D
Cancer letters. 2021;:13-25
Abstract
Proteasome inhibitors (PIs), used in the treatment of plasma cell myeloma (PCM), interfere with the degradation of misfolded proteins leading to activation of unfolded protein response (UPR) and cell death. However, despite initial strong antimyeloma effects, PCM cells eventually develop acquired resistance to PIs. The pleiotropic role of ʟ-glutamine (Gln) in cellular functions makes inhibition of Gln metabolism a potentially good candidate for combination therapy. Here, we show that PCM cells, both sensitive and resistant to PIs, express membrane Gln transporter (ASCT2), require extracellular Gln for survival, and are sensitive to ASCT2 inhibitors (ASCT2i). ASCT2i synergistically potentiate the cytotoxic activity of PIs by inducing apoptosis and modulating autophagy. Combination of ASCT2 inhibitor V9302 and proteasome inhibitor carfilzomib upregulates the intracellular levels of ROS and oxidative stress markers and triggers catastrophic UPR as shown by upregulated spliced Xbp1 mRNA, ATF3 and CHOP levels. Moreover, analysis of RNA sequencing revealed that the PI in combination with ASCT2i reduced the levels of Gln metabolism regulators such as MYC and NRAS. Analysis of PCM patients' data revealed that upregulated ASCT2 and other Gln metabolism regulators are associated with advanced disease stage and with PIs resistance. Altogether, we identified a potent therapeutic approach that may prevent acquired resistance to PIs and may contribute to the improvement of treatment of patients suffering from PCM.
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Recapitulation of selective nuclear import and export with a perfectly repeated 12mer GLFG peptide.
Ng, SC, Güttler, T, Görlich, D
Nature communications. 2021;(1):4047
Abstract
The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules while allowing facilitated passage of importins and exportins, which in turn shuttle cargo into or out of cell nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains. NPCs contain several distinct FG domains, each comprising variable repeats. Nevertheless, we now found that sequence heterogeneity is no fundamental requirement for barrier function. Instead, we succeeded in engineering a perfectly repeated 12mer GLFG peptide that self-assembles into a barrier of exquisite transport selectivity and fast transport kinetics. This barrier recapitulates RanGTPase-controlled importin- and exportin-mediated cargo transport and thus represents an ultimately simplified experimental model system. An alternative proline-free sequence forms an amyloid FG phase. Finally, we discovered that FG phases stain bright with 'DNA-specific' DAPI/ Hoechst probes, and that such dyes allow for a photo-induced block of nuclear transport.
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Safety and Efficacy of Human Chorionic Gonadotropin Hormone-Derivative EA-230 in Cardiac Surgery Patients: A Randomized Double-Blind Placebo-Controlled Study.
van Groenendael, R, Beunders, R, Hemelaar, P, Hofland, J, Morshuis, WJ, van der Hoeven, JG, Gerretsen, J, Wensvoort, G, Kooistra, EJ, Claassen, WJ, et al
Critical care medicine. 2021;(5):790-803
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Abstract
OBJECTIVES To determine the safety and efficacy of human chorionic gonadotropin hormone-derivative EA-230 in cardiac surgery patients. Cardiac surgery induces systemic inflammation and may impair renal function, affecting patient outcome. EA-230 exerted immunomodulatory and renoprotective effects in preclinical models and was safe and showed efficacy in phase I and II human studies. DESIGN Double-blinded, placebo-controlled, randomized study. SETTING Collaboration of the Cardiothoracic Surgery, Anesthesiology, and the Intensive Care departments of a tertiary hospital in the Netherlands. PATIENTS One hundred eighty patients undergoing an on-pump coronary artery bypass procedure with or without concomitant valve surgery. INTERVENTIONS Ninety mg/kg/hr EA-230 or placebo administered during surgery. MEASUREMENTS AND MAIN RESULTS During the study, no safety concerns emerged. EA-230 did not modulate interleukin-6 plasma concentrations (area under the curve 2,730 pg/mL × hr [1,968-3,760] vs 2,680 pg/mL × hr [2,090-3,570] for EA-230 and placebo group, respectively; p = 0.80). Glomerular filtration rate increased following surgery (mean ± sem increase in the EA-230 vs placebo groups: glomerular filtration rateiohexol measured using iohexol plasma clearance: 19 ± 2 vs 16 ± 2 mL/min/1.73 m2; p = 0.13 and estimated glomerular filtration rate with the Modification of Diet in Renal Disease equation using creatinine: 6 ± 1 vs 2 ± 1 mL/min/1.73 m2; p = 0.01). The "injury" stage of the Risk, Injury, Failure, Loss of kidney function, and End-stage kidney disease criteria for acute kidney injury was 7% in the EA-230 group versus 18% in the placebo group (p = 0.07). In addition, EA-230-treated patients had a less positive fluid balance compared with placebo-treated patients (217 ± 108 vs 605 ± 103 mL; p = 0.01), while the use of vasoactive agents was similar in both groups (p = 0.39). Finally, hospital length of stay was shorter in EA-230 treated patients (8 d [7-11] vs 10 d [8-12]; p = 0.001). Efficacy results were more pronounced in patients that had longer duration of surgery and thus longer duration of study drug infusion. CONCLUSIONS EA-230 was safe in patients undergoing on-pump cardiac surgery. It did not modulate interleukin-6 plasma concentrations but appeared to exert beneficial renal and cardiovascular effects and shortened in-hospital length of stay.
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Monitoring urinary collagen metabolite changes following collagen peptide ingestion and physical activity using ELISA with anti active collagen oligopeptide antibody.
Osawa, Y, Nomura, K, Kimira, Y, Kushibe, S, Takeyama, KI, Nagao, M, Kataoka-Matsushita, A, Koizumi, S, Mano, H
Scientific reports. 2021;(1):13527
Abstract
Active collagen oligopeptides (ACOP) are bioactive collagen-derived peptides detected by a recently-established ELISA. To facilitate studies of the function and metabolism of these products, this study aims to determine which of these peptides is recognized by a novel anti-ACOP antibody used in this ELISA. We then investigate the effect of collagen peptide (CP) ingestion and exercise on urinary ACOP concentrations in a cohort of university student athletes using colorimetric, LC-MS/MS, and ELISA. We observed that the antibody showed strong cross-reactivity to Pro-Hyp and Gly-Pro-Hyp and weak cross-reactivity to commercial CP. CP ingestion increased the urinary level of ACOP over time, which correlated highly with urinary levels of peptide forms of Hyp and Pro-Hyp. Physical activity significantly decreased the urinary ACOP level. This study demonstrates changes in urinary ACOP following oral CP intake and physical activity using ELISA with the novel anti-ACOP antibody. Thus, ACOP may be useful as a new biomarker for collagen metabolism.