N-acetyl-aspartate metabolism at the interface of cancer, immunity, and neurodegeneration.

N-acetyl-L-aspartic acid (NAA) is a prominent amino acid derivative primarily associated with vertebrate brain metabolism. This review delineates the critical role of NAA across various cell types and its significance in pathophysiological contexts, including Canavan disease and cancer metabolism. Although traditionally linked with myelination and aspartoacylase-driven carbon donation, its significance as a carbon source for myelination remains debated. Evidence suggests that intact NAA might substantially impact cellular signaling, particularly processes such as histone acetylation. Beyond the brain, NAA metabolism's relevance is evident in diverse tissues, such as adipocytes, immune cells, and notably, cancer cells. In several cancer types, there is an observed upregulation of NAA synthesis accompanied by a simultaneous downregulation of its degradation. This pattern highlights the potential signaling role of intact NAA in disease.

Utility of Three Flow Imaging Microscopy Instruments for Image Analysis in Evaluating four Types of Subvisible Particle in Biopharmaceuticals.

Subvisible particles (SVPs) are a critical quality attribute of parenteral and ophthalmic products. United States Pharmacopeia recommends the characterizations of SVPs which are classified into intrinsic, extrinsic, and inherent particles. Flow imaging microscopy (FIM) is useful as an orthogonal method in both the quantification and classification of SVPs because FIM instruments provide particle images. In addition to the conventionally used FlowCam (Yokogawa Fluid Imaging Technologies) and Micro-Flow Imaging (Bio-Techne) instruments, the iSpect DIA-10 (Shimadzu) instrument has recently been released. The three instruments have similar detection principles but different optical settings and image processing, which may lead to different results of the quantification and classification of SVPs based on the information from particle images. The present study compares four types of SVP (protein aggregates, silicone oil droplets, and surrogates for solid free-fatty-acid particles, milled-lipid particles, and sprayed-lipid particles) to compare the results of size distributions and classification abilities obtained using morphological features and a deep-learning approach. Although the three FIM instruments were effective in classifying the four types of SVP through convolutional neural network analysis, there was no agreement on the size distribution for the same protein aggregate solution, suggesting that using the classifiers of the FIM instruments could result in different evaluations of SVPs in the field of biopharmaceuticals.

Towards quantification and differentiation of protein aggregates and silicone oil droplets in the low micrometer and submicrometer size range by using oil-immersion flow imaging microscopy and convolutional neural networks.

Biopharmaceutical product characterization benefits from the quantification and differentiation of unwanted protein aggregates and silicone oil droplets to support risk assessment and control strategies as part of the development. Flow imaging microscopy is successfully applied to differentiate the two impurities in the size range larger than about 5 µm based on their morphological appearance. In our study we applied the combination of oil-immersion flow imaging microscopy and convolutional neural networks to extend the size range below 5 µm. It allowed to differentiate and quantify heat stressed therapeutic monoclonal antibody aggregates from artificially generated silicone oil droplets with misclassification rates of about 10% in the size range between 0.3 and 5 µm. By comparing the misclassifications across the tested size range, particles in the low submicron size range were particularly difficult to differentiate as their morphological appearance becomes very similar.

Oil-Immersion Flow Imaging Microscopy for Quantification and Morphological Characterization of Submicron Particles in Biopharmaceuticals.

Flow imaging microscopy (FIM) is widely used to analyze subvisible particles starting from 2 µm in biopharmaceuticals. Recently, an oil-immersion FIM system emerged, the FlowCam Nano, designed to enable the characterization of particle sizes even below 2 µm. The aim of our study was to evaluate oil-immersion FIM (by using FlowCam Nano) in comparison to microfluidic resistive pulse sensing and resonant mass measurement for sizing and counting of particles in the submicron range. Polystyrene beads, a heat-stressed monoclonal antibody formulation and a silicone oil emulsion, were measured to assess the performance on biopharmaceutical relevant samples, as well as the ability to distinguish particle types based on instrument-derived morphological parameters. The determination of particle sizes and morphologies suffers from inaccuracies due to a low image contrast of small particles and light-scattering effects. The ill-defined measured volume impairs an accurate concentration determination. Nevertheless, FlowCam Nano in its current design complements the limited toolbox of submicron particle analysis of biopharmaceuticals by providing particle images in a size range that was previously not accessible with commercial FIM instruments.

Fructose Metabolism in Cancer.

The interest in fructose metabolism is based on the observation that an increased dietary fructose consumption leads to an increased risk of obesity and metabolic syndrome. In particular, obesity is a known risk factor to develop many types of cancer and there is clinical and experimental evidence that an increased fructose intake promotes cancer growth. The precise mechanism, however, in which fructose induces tumor growth is still not fully understood. In this article, we present an overview of the metabolic pathways that utilize fructose and how fructose metabolism can sustain cancer cell proliferation. Although the degradation of fructose shares many of the enzymes and metabolic intermediates with glucose metabolism through glycolysis, glucose and fructose are metabolized differently. We describe the different metabolic fates of fructose carbons and how they are connected to lipogenesis and nucleotide synthesis. In addition, we discuss how the endogenous production of fructose from glucose via the polyol pathway can be beneficial for cancer cells.

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2.

A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.

Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans.

Variant nomenclature: the variants were made in the NorB subunit if not indicated by the superscript c, which are variants in the NorC subunit (e.g. E122A = exchange of Glu-122 in NorB for an Ala, E71cD; exchange of Glu-71 in NorC for an Asp). Bacterial NO reductases (NORs) are integral membrane proteins from the heme-copper oxidase superfamily. Most heme-copper oxidases are proton-pumping enzymes that reduce O2 as the last step in the respiratory chain. With electrons from cytochrome c, NO reductase (cNOR) from Paracoccus (P.) denitrificans reduces NO to N2O via the following reaction: 2NO+2e-+2H+→N2O+H2O. Although this reaction is as exergonic as O2-reduction, cNOR does not contribute to the electrochemical gradient over the membrane. This means that cNOR does not pump protons and that the protons needed for the reaction are taken from the periplasmic side of the membrane (since the electrons are donated from this side). We previously showed that the P. denitrificans cNOR uses a single defined proton pathway with residues Glu-58 and Lys-54 from the NorC subunit at the entrance. Here we further strengthened the evidence in support of this pathway. Our further aim was to define the continuation of the pathway and the immediate proton donor for the active site. To this end, we investigated the region around the calcium-binding site and both propionates of heme b3 by site directed mutagenesis. Changing single amino acids in these areas often had severe effects on cNOR function, with many variants having a perturbed active site, making detailed analysis of proton transfer properties difficult. Our data does however indicate that the calcium ligation sphere and the region around the heme b3 propionates are important for proton transfer and presumably contain the proton donor. The possible evolutionary link between the area for the immediate donor in cNOR and the proton loading site (PLS) for pumped protons in oxygen-reducing heme-copper oxidases is discussed.

The nitric-oxide reductase from Paracoccus denitrificans uses a single specific proton pathway.

The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

Structural differences between the closed and open states of channelrhodopsin-2 as observed by EPR spectroscopy.

Channelrhodopsin is a cation channel with the unique property of being activated by light. To address structural changes of the open state of the channel, two variants, which contain either 1 or 2 wild-type cysteines, were derivatised with nitroxide spin label and subjected to electron paramagnetic resonance spectroscopy. Both variants contained the C128T mutation to trap the long-lived P3(520) state by illumination. Comparison of spin-spin distances in the dark state and after illumination reflect conformational changes in the conductive P3(520) state involving helices B and F. Spin distance measurements reveal that channelrhodopsin forms a dimer even in the absence of intermolecular N-terminal cysteines.

Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating.

The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements. We have resolved the vibrational changes associated with the open states of the channel (P(2)(390) and P(3)(520)) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane α-helices with a t(1/2) = 60 µs, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t(1/2) = 10 µs), with the latter being reprotonated by aspartic acid 156 (t(1/2) = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. Previous conclusions on the involvement of glutamic acid 90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the nonconductive P(4)(480) state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions and the protein conformational changes to the gating of the cation channel.

Proton transfer in the quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus during reduction of oxygen.

Bacterial nitric oxide reductases (NOR) are integral membrane proteins that catalyse the reduction of nitric oxide to nitrous oxide, often as a step in the process of denitrification. Most functional data has been obtained with NORs that receive their electrons from a soluble cytochrome c in the periplasm and are hence termed cNOR. Very recently, the structure of a different type of NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246]. In this study, we have investigated the reaction between this qNOR and oxygen. Our results show that, like some cNORs, the G. stearothermophilus qNOR is capable of O(2) reduction with a turnover of ~3electronss(-1) at 40°C. Furthermore, using the so-called flow-flash technique, we show that the fully reduced (with three available electrons) qNOR reacts with oxygen in a reaction with a time constant of 1.8ms that oxidises the low-spin heme b. This reaction is coupled to proton uptake from solution and presumably forms a ferryl intermediate at the active site. The pH dependence of the reaction is markedly different from a corresponding reaction in cNOR from Paracoccus denitrificans, indicating that possibly the proton uptake mechanism and/or pathway differs between qNOR and cNOR. This study furthermore forms the basis for investigation of the proton transfer pathway in qNOR using both variants with putative proton transfer elements modified and measurements of the vectorial nature of the proton transfer. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).