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Molecular Pharmaceutics Jun 2024Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for...
Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for subcutaneous administration. Understanding self-association mechanisms is therefore critical to the design and selection of candidates with acceptable developability to advance to clinical trials. The combination of experiments and in silico modeling presents a powerful tool to elucidate the interface of self-association. RSA of monoclonal antibodies has been studied extensively under different solution conditions and have been shown to involve interactions for both the antigen-binding fragment and the crystallizable fragment. Novel modalities such as bispecific antibodies, antigen-binding fragments, single-chain-variable fragments, and diabodies constitute a fast-growing class of antibody-based therapeutics that have unique physiochemical properties compared to monoclonal antibodies. In this study, the RSA interface of a diabody-interleukin 22 fusion protein (FP-1) was studied using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) in combination with in silico modeling. Taken together, the results show that a complex solution behavior underlies the self-association of FP-1 and that the interface thereof can be attributed to a specific segment in the variable light chain of the diabody. These findings also demonstrate that the combination of HDX-MS with in silico modeling is a powerful tool to guide the design and candidate selection of novel biotherapeutic modalities.
PubMed: 38922328
DOI: 10.1021/acs.molpharmaceut.4c00169 -
Journal of the American Chemical Society Jun 2024The receptor for advanced glycation end products (RAGE) plays a crucial role in inflammation-related pathways and various chronic diseases. Despite the recognized...
The receptor for advanced glycation end products (RAGE) plays a crucial role in inflammation-related pathways and various chronic diseases. Despite the recognized significance of N-glycosylation in the ligand-binding V domain (VD) of RAGE, a comprehensive understanding of the site-activity and structure-activity relationships is lacking due to the challenges in obtaining homogeneous glycoprotein samples through biological expression. Here, we combined chemical and chemoenzymatic approaches to synthesize RAGE-VD and its congeners with Asn3-glycosylation by incorporating precise N-glycan structures. Evaluation of these samples revealed that, in comparison to other RAGE-VD forms, α2,6-sialylated N-glycosylation at the Asn3 site results in more potent inhibition of HMGB1-induced nuclear factor-κB (NF-κB) expression in RAGE-overexpressing cells. Hydrogen/deuterium exchange-mass spectrum analysis revealed a sialylated RAGE-VD-induced interaction region within HMGB1. Conversely, Asn3 N-glycosylation in VD has negligible effects on RAGE-VD/S100B interactions. This study established an approach for accessing homogeneously glycosylated RAGE-VD and explored the modulatory effects of N-glycosylation on the interactions between RAGE-VD and its ligand proteins.
PubMed: 38917169
DOI: 10.1021/jacs.4c01413 -
Dalton Transactions (Cambridge, England... Jun 2024The coordination chemistry of an amine-rich Cp ligand has been explored with cobalt. We demonstrate that in the presence of NaCo(CO), the cationic precursor [CpN3]+...
The coordination chemistry of an amine-rich Cp ligand has been explored with cobalt. We demonstrate that in the presence of NaCo(CO), the cationic precursor [CpN3]+ yields the complex CpN3CoI(CO)2. While 2e oxidation generates new Co complexes such as [CpN3Co(NCMe)3]2+ and CpN3CoI2(CO), subsequent ligand loss is facile, generating free [CpN3]+ or the protonated dication [CpN3H]2+. We have structurally characterized both these ligand release products single crystal X-ray diffraction and obtained thermochemical C-H bond strengths experiment and density functional theory (DFT). Upon reversible 1e reduction, the radical cation [CpN3H]˙+ has a weak C-H BDFE of 52 kcal mol in acetonitrile. Mechanistic analysis shows that [CpN3H]˙+ undergoes radical-radical disproportionation in the absence of exogenous H-atom acceptors, which is supported by deuterium isotope labelling experiments. Structural comparison of these organic molecules shows a high degree of iminium-like electron delocalization over the C-N bonds connected to the central five-membered ring.
PubMed: 38916110
DOI: 10.1039/d4dt01560f -
The Journal of Experimental Biology Jun 2024Black soldier fly (Hermetia illucens) larvae are used to upcycle biowaste into insect biomass for animal feed. Previous research on black soldier fly has explored the...
Black soldier fly (Hermetia illucens) larvae are used to upcycle biowaste into insect biomass for animal feed. Previous research on black soldier fly has explored the assimilation of dietary fatty acids (FAs), but endogenous FA synthesis and modification remain comparatively unexplored. This study presents a 1H/2H-NMR methodology for measuring lipid synthesis in black soldier fly larvae using diluted deuterated water (2H2O) as a stable isotopic tracer delivered through the feeding media. This approach was validated by measuring 2H incorporation into the larvae's body water and consequent labelling of FA esterified into triacylglycerols. A 5% 2H enrichment in the body water, adequate to label the FA, is achieved after 24 h in a substrate with 10% 2H2O. A standard feeding trial using an invasive macroalgae was designed to test this method, revealing de novo lipogenesis was lower in larvae fed with macroalgae, probably related to the poor nutritional value of the diet.
Topics: Animals; Larva; Seaweed; Deuterium Oxide; Magnetic Resonance Spectroscopy; Animal Feed; Fatty Acids; Lipids; Diptera; Simuliidae; Diet
PubMed: 38916067
DOI: 10.1242/jeb.247941 -
BioRxiv : the Preprint Server For... Jun 2024The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires...
The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires the cofactor Nub1, yet the underlying mechanisms remain unknown. Here, we reconstituted a minimal system and revealed that Nub1 utilizes FAT10's intrinsic instability to trap its N-terminal ubiquitin-like domain in an unfolded state and deliver it to the 26S proteasome for engagement, allowing the degradation of FAT10-ylated substrates in a ubiquitin- and p97-independent manner. Through hydrogen-deuterium exchange, structural modeling, and site-directed mutagenesis, we identified the formation of a peculiar complex with FAT10 that activates Nub1 for docking to the 26S proteasome, and our cryo-EM studies visualized the highly dynamic Nub1 complex bound to the proteasomal Rpn1 subunit during FAT10 delivery and the early stages of ATP-dependent degradation. These studies thus identified a novel mode of cofactor-mediated, ubiquitin-independent substrate delivery to the 26S proteasome that relies on trapping partially unfolded states for engagement by the proteasomal ATPase motor.
PubMed: 38915702
DOI: 10.1101/2024.06.12.598715 -
The Journal of Organic Chemistry Jun 2024Enamines are difficult to prepare on the bench due to their instability, which results in side reactions, decompositions, poor yields, etc. Herein, we developed a simple...
Enamines are difficult to prepare on the bench due to their instability, which results in side reactions, decompositions, poor yields, etc. Herein, we developed a simple and effective method for making bench-stable enamines using a very low amount of nickel catalyst loading. The deuterium exchange, competitive reaction, and radical clock experiment have all been found to favor the ionic mechanism of this alkene isomerization. Scale-up and [3 + 2] annulation reaction of enamines with activated cyclopropane to deliver cyclopentane derivatives have shown the value of this method in organic synthesis.
PubMed: 38912727
DOI: 10.1021/acs.joc.4c00154 -
RSC Medicinal Chemistry Jun 20243,3'-Diselenodipropionic acid (DSePA), a selenocystine derivative, has been previously reported as an oral supplement for anticancer/radio-modulation activities. The...
3,3'-Diselenodipropionic acid (DSePA), a selenocystine derivative, has been previously reported as an oral supplement for anticancer/radio-modulation activities. The present study is focused on devising a strategy to synthesize and characterize the deuterated derivative of DSePA and on understanding the effect of deuteration on its therapeutic index by comparing its cytotoxicity in cancerous non-cancerous cell types. In this context, the synthesis of 3,3'-diselenodipropionic acid-D (D-DSePA) was accomplished in ∼42% yield. Further, the results clearly established that the deuteration of DSePA significantly reduced its cytotoxicity in non-cancerous cell types while retaining its cytotoxicity in cancerous cell lines. Together, D-DSePA displayed a ∼5-fold higher therapeutic index than the non-deuterated derivative for anticancer activity. The biochemical and NMR studies confirmed that the better biocompatibility of D-DSePA than its non-deuterated derivative in non-cancerous cells was due to its ability to undergo slower redox reactions and to cause lesser inhibition of intracellular redox enzymes.
PubMed: 38911162
DOI: 10.1039/d4md00105b -
Physical Review Letters Jun 2024In this study, we discovered a turbulence transition in a large helical device. The turbulence level and turbulence-driven energy transport decrease to a specific...
In this study, we discovered a turbulence transition in a large helical device. The turbulence level and turbulence-driven energy transport decrease to a specific transition density and increase above it. The ruling turbulences below and above the transition density were ion-temperature gradient (ITG) and resistive-interchange (RI) turbulences, consistent with the predictions of gyrokinetic theory and two-fluid MHD model, respectively. Isotope experiments on hydrogen (H) and deuterium (D) clarified the role of transitions. In the ITG regime, turbulence levels and energy transport were comparable in the H and D plasmas. In contrast, in the RI regime, they were clearly suppressed in the D plasma. The results provide crucial knowledge for understanding isotope effects and future optimization of stellarator and heliotron devices.
PubMed: 38905665
DOI: 10.1103/PhysRevLett.132.235101 -
Proceedings of the National Academy of... Jun 2024Hydrogen isotope ratios (δH) represent an important natural tracer of metabolic processes, but quantitative models of processes controlling H-fractionation in aquatic...
Hydrogen isotope ratios (δH) represent an important natural tracer of metabolic processes, but quantitative models of processes controlling H-fractionation in aquatic photosynthetic organisms are lacking. Here, we elucidate the underlying physiological controls of H/H fractionation in algal lipids by systematically manipulating temperature, light, and CO(aq) in continuous cultures of the haptophyte . We analyze the hydrogen isotope fractionation in alkenones (α), a class of acyl lipids specific to this species and other haptophyte algae. We find a strong decrease in the α with increasing CO(aq) and confirm α correlates with temperature and light. Based on the known biosynthesis pathways, we develop a cellular model of the δH of algal acyl lipids to evaluate processes contributing to these controls on fractionation. Simulations show that longer residence times of NADPH in the chloroplast favor a greater exchange of NADPH with H-richer intracellular water, increasing α. Higher chloroplast CO(aq) and temperature shorten NADPH residence time by enhancing the carbon fixation and lipid synthesis rates. The inverse correlation of α to CO(aq) in our cultures suggests that carbon concentrating mechanisms (CCM) do not achieve a constant saturation of CO at the Rubisco site, but rather that chloroplast CO varies with external CO(aq). The pervasive inverse correlation of α with CO(aq) in the modern and preindustrial ocean also suggests that natural populations may not attain a constant saturation of Rubisco with the CCM. Rather than reconstructing growth water, α may be a powerful tool to elucidate the carbon limitation of photosynthesis.
Topics: Carbon Dioxide; Haptophyta; Lipids; Photosynthesis; Hydrogen; Chloroplasts; Deuterium; NADP; Temperature; Chemical Fractionation; Lipid Metabolism
PubMed: 38905238
DOI: 10.1073/pnas.2318570121 -
Physical Chemistry Chemical Physics :... Jun 2024The Zr-2.5Nb alloy is a typical pressure tube material in heavy water nuclear reactors, and an increase of hydrogen isotope content in the alloy during service can pose...
The Zr-2.5Nb alloy is a typical pressure tube material in heavy water nuclear reactors, and an increase of hydrogen isotope content in the alloy during service can pose major safety risks; hot vacuum extraction-mass spectrometry is an efficient method for evaluating hydrogen isotope concentrations in the Zr-2.5Nb alloy. This work investigates the kinetics and thermodynamic properties of deuterium (D) absorption and desorption of the Zr-2.5Nb alloy using the constant volume adsorption method and the hot vacuum extraction method. In addition to the previously reported volume contraction model, it was observed that at 600 °C and above, the reaction between D and Zr-2.5Nb is dominated by diffusion, while the reaction is predominantly influenced by surface adsorption and dissociation below 600 °C. Phase transition sequence of Zr-2.5Nb deuterides during non-isothermal desorption was established using quantitatively calibrated thermal desorption spectra combined with the phase transition process of deuteride decomposition. These results can provide important references for optimizing the process parameters of the hot vacuum extraction-mass spectrometry method.
PubMed: 38904087
DOI: 10.1039/d4cp01512f