Polysulfur-based bulking of dynamin-related protein 1 prevents ischemic sulfide catabolism and heart failure in mice.

The presence of redox-active molecules containing catenated sulfur atoms (supersulfides) in living organisms has led to a review of the concepts of redox biology and its translational strategy. Glutathione (GSH) is the body's primary detoxifier and antioxidant, and its oxidized form (GSSG) has been considered as a marker of oxidative status. However, we report that GSSG, but not reduced GSH, prevents ischemic supersulfide catabolism-associated heart failure in male mice by electrophilic modification of dynamin-related protein (Drp1).

Isolation method of brain microvessels from small frozen human brain tissue for blood-brain barrier protein expression analysis.

Background: Protein expression analysis of isolated brain microvessels provides valuable insights into the function of the blood-brain barrier (BBB). However, isolation of brain microvessels from human brain tissue, particularly in small quantities, poses significant challenges. This study presents a method for isolating brain microvessels from a small amount of frozen human brain tissue, adapting techniques from an established mouse brain capillary isolation method.

Progressive Amyloid-β Accumulation in the Brain leads to Altered Protein Expressions in the Liver and Kidneys of APP knock-in Mice.

Impaired hepatic and renal function influence Alzheimer's disease (AD) progression; however, whether AD progression affects these important organ functions remains unclear. Here, we investigated the impact of AD progression, characterized by brain amyloid-beta (Aβ) accumulation, on liver and kidney function of App (APP-KI) mice using quantitative proteomics. SWATH-based quantitative proteomics revealed changes in mitochondrial, drug metabolism, and pharmacokinetic-related proteins in mouse liver and kidneys during the early (2-month-old) and intermediate (5-month-old) stages of Aβ accumulation.

Supersulfide formation in the sinus mucosa of chronic rhinosinusitis.

Objectives: Disruption of the oxidative stress defense system is involved in developing various diseases. Sulfur compounds such as glutathione (GSH) and cysteine (CysSH) are representative antioxidants in the body. Recently, supersulfides, including reactive persulfide and polysulfide species, have gained attention as potent antioxidants regulating oxidative stress and redox signaling.

New aspects of redox signaling mediated by supersulfides in health and disease.

Oxygen molecules accept electrons from the respiratory chain in the mitochondria and are responsible for energy production in aerobic organisms. The reactive oxygen species formed via these oxygen reduction processes undergo complicated electron transfer reactions with other biological substances, which leads to alterations in their physiological functions and cause diverse biological and pathophysiological consequences (e.g.

2H-Thiopyran-2-thione sulfine, a compound for converting HS to HSOH/HS and increasing intracellular sulfane sulfur levels.

Reactive sulfane sulfur species such as persulfides (RSSH) and HS are important redox regulators and closely linked to HS signaling. However, the study of these species is still challenging due to their instability, high reactivity, and the lack of suitable donors to produce them. Herein we report a unique compound, 2H-thiopyran-2-thione sulfine (TTS), which can specifically convert HS to HSOH, and then to HS in the presence of excess HS.

Longevity control by supersulfide-mediated mitochondrial respiration and regulation of protein quality.

Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics.

Proteomic alterations in the brain and blood-brain barrier during brain Aβ accumulation in an APP knock-in mouse model of Alzheimer's disease.

Background: Blood-brain barrier (BBB) dysfunction is supposed to be an early event in the development of Alzheimer's disease (AD). This study aimed to investigate the relationship between BBB alterations and AD progression in terms of amyloid-β peptide (Aβ) accumulation in the brains of humanized amyloid precursor protein knock-in (APP-KI) mice.

Methods: Brain Aβ accumulation was examined using immunohistochemical analysis.

Supersulfide catalysis for nitric oxide and aldehyde metabolism.

Abundant formation of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiols and proteins (supersulfidation), has been observed. We found here that supersulfides catalyze -nitrosoglutathione (GSNO) metabolism via glutathione-dependent electron transfer from aldehydes by exploiting alcohol dehydrogenase 5 (ADH5). ADH5 is a highly conserved bifunctional enzyme serving as GSNO reductase (GSNOR) that down-regulates NO signaling and formaldehyde dehydrogenase (FDH) that detoxifies formaldehyde in the form of glutathione hemithioacetal.

Persulfide Biosynthesis Conserved Evolutionarily in All Organisms.

Persulfides/polysulfides are sulfur-catenated molecular species (, R-S-R',  > 2; R-S-H,  > 1, with R = cysteine, glutathione, and proteins), such as cysteine persulfide (CysSSH). These species are abundantly formed as endogenous metabolites in mammalian and human cells and tissues. However, the persulfide synthesis mechanism has yet to be thoroughly discussed.

Supersulphides provide airway protection in viral and chronic lung diseases.

Supersulphides are inorganic and organic sulphides with sulphur catenation with diverse physiological functions. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulphide synthase (CPERS). Here, we identify protective functions of supersulphides in viral airway infections (influenza and COVID-19), in aged lungs and in chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF).

Development of a method for isolating brain capillaries from a single neonatal mouse brain and comparison of proteomic profiles between neonatal and adult brain capillaries.

Background: The functions and protein expressions of the blood-brain barrier are changed throughout brain development following birth. This study aimed to develop a method to isolate brain capillaries from a single frozen neonatal mouse brain and elucidate the enrichment of brain capillaries by quantitative proteomic analysis. We further compared the expression profile of proteins between neonatal and adult brain capillary fractions.

Influence of MDR1 gene polymorphism (2677G>T) on expression and function of P-glycoprotein at the blood-brain barrier: utilizing novel P-glycoprotein humanized mice with mutation.

P-glycoprotein, the encoded product of the MDR1 / ABCB1 gene in humans, is expressed in numerous tissues including brain capillary endothelial cells and restricts the distribution of xenobiotics into the brain as an efflux pump. Although a large number of single nucleotide polymorphisms in the MDR1 gene have been identified, the influence of the nonsynonymous 2677G>T/A single nucleotide polymorphism on P-glycoprotein at the blood-brain barrier has remained unclear. In the present study, we developed a novel P-glycoprotein humanized mouse line carrying the 2677G>T mutation by utilizing a mouse artificial chromosome vector constructed by genetic engineering technology and we evaluated the influence of 2677G>T on the expression and function of P-glycoprotein at the blood-brain barrier in vivo .

Diurnal Changes in Protein Expression at the Blood-Brain Barrier in Mice.

Circadian rhythms influence the transport function of the blood-brain barrier (BBB) and peripheral organs. However, the influence of circadian rhythms on protein expression in the BBB remains to be completely elucidated. Therefore, we aimed to investigate diurnal changes in protein expression in the mouse BBB using quantitative proteomics.

Targeted proteomics for cancer biomarker verification and validation.

Targeted proteomics is a method that measures the amount of target proteins via liquid chromatography-tandem mass spectrometry and is used to verify and validate the candidate cancer biomarker proteins. Compared with antibody-based quantification methods such as ELISA, targeted proteomics enables rapid method development, simultaneous measurement of multiple proteins, and high-specificity detection of modifications. Moreover, by spiking the internal standard peptide, targeted proteomics detects the absolute amounts of marker proteins, which is essential for determining the cut-off values for diagnosis and thus for multi-institutional validation.

Knockdown of Podocalyxin Post-Transcriptionally Induces the Expression and Activity of ABCB1/MDR1 in Human Brain Microvascular Endothelial Cells.

Podocalyxin (PODXL) is a highly sialylated transmembrane protein that is expressed on the luminal membrane of brain microvascular endothelial cells. To clarify the role of PODXL in the blood-brain barrier (BBB), the present study aimed to investigate the effect of PODXL-knockdown on protein expression, especially the expression of ABCB1/MDR1, in human microvascular endothelial cells (hCMEC/D3). By quantitative proteomics, gene ontology enrichment with differentially expressed proteins showed that PODXL-knockdown influenced the immune response and intracellular trafficking.

Acetylation of the influenza A virus polymerase subunit PA in the N-terminal domain positively regulates its endonuclease activity.

The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity.

Oral Coadministration of Zn-Insulin with d-Form Small Intestine-Permeable Cyclic Peptide Enhances Its Blood Glucose-Lowering Effect in Mice.

Oral delivery of insulin remains a challenge owing to its poor permeability across the small intestine and enzymatic digestion in the gastrointestinal tract. In a previous study, we identified a small intestine-permeable cyclic peptide, C-DNPGNET-C (C-C disulfide bond, cyclic DNP peptide), which facilitated the permeation of macromolecules. Here, we showed that intraintestinal and oral coadministration of insulin with the cyclic DNP derivative significantly reduced blood glucose levels by increasing the portal plasma insulin concentration following permeation across the small intestine of mice.

Proteomic Evaluation of Plasma Membrane Fraction Prepared from a Mouse Liver and Kidney Using a Bead Homogenizer: Enrichment of Drug-Related Transporter Proteins.

Quantifying the protein levels of drug transporters in plasma membrane fraction helps elucidate the function of these transporters. In this study, we conducted a proteomic evaluation of enriched drug-related transporter proteins in plasma membrane fraction prepared from mouse liver and kidney tissues using the membrane protein extraction kit and a bead homogenizer. Crude and plasma membrane fractions were prepared using either the Dounce or bead homogenizer, and protein levels were determined using quantitative proteomics.

Efficient isolation of brain capillary from a single frozen mouse brain for protein expression analysis.

Isolated brain capillaries are essential for analyzing the changes of protein expressions at the blood-brain barrier (BBB) under pathological conditions. The standard brain capillary isolation methods require the use of at least five mouse brains in order to obtain a sufficient amount and purity of brain capillaries. The purpose of this study was to establish a brain capillary isolation method from a single mouse brain for protein expression analysis.

Identification of Cell-Surface Proteins Endocytosed by Human Brain Microvascular Endothelial Cells In Vitro.

Cell-surface proteins that can endocytose into brain microvascular endothelial cells serve as promising candidates for receptor-mediated transcytosis across the blood-brain barrier (BBB). Here, we comprehensively screened endocytic cell-surface proteins in hCMEC/D3 cells, a model of human brain microvascular endothelial cells, using surface biotinylation methodology and sequential window acquisition of all theoretical fragment-ion spectra-mass spectrometry (SWATH-MS)-based quantitative proteomics. Using this method, we identified 125 endocytic cell-surface proteins from hCMEC/D3 cells.

Changes of Blood-Brain Barrier and Brain Parenchymal Protein Expression Levels of Mice under Different Insulin-Resistance Conditions Induced by High-Fat Diet.

Purpose: The purpose of the present study was to investigate changes of blood-brain barrier (BBB) and brain parenchymal protein expression due to type II diabetes mellitus (T2DM) induced by a high-fat diet (HFD) by using SWATH-based quantitative proteomics.

Methods: Mice were fed a HFD for 2 or 10 weeks, and then SWATH-based quantitative proteomic analysis, western blot analysis, immunohistochemistry and functional transport studies were performed.

Results: In brain capillaries, expression levels of BBB transporters (Glut1, P-glycoprotein) and tight-junction proteins (claudin-5, occludin) were significantly reduced in HFD mice at 2 weeks, but recovered to the levels in the normal diet (ND) group at 10 weeks.

Large-Scale Quantitative Comparison of Plasma Transmembrane Proteins between Two Human Blood-Brain Barrier Model Cell Lines, hCMEC/D3 and HBMEC/ciβ.

Transmembrane (TM) proteins localized at the plasma membrane, such as transporters and receptors, play important roles in regulating the selective permeability of the blood-brain barrier (BBB). The purpose of the present study was to clarify the differences in the expression levels of TM proteins in the plasma membrane between two established human BBB model cell lines, hCMEC/D3 and HBMEC/ciβ, in order to assist researchers in selecting the most appropriate cell line for particular purposes. We first confirmed that plasma membranes could be enriched sufficiently for a quantitative proteomics study by using the Plasma Membrane Protein Extraction Kit provided by BioVision with a modified protocol.

Knockdown of Orphan Transporter SLC22A18 Impairs Lipid Metabolism and Increases Invasiveness of HepG2 Cells.

Purpose: The aim of this work is to investigate the roles of solute carrier family 22 member 18 (SLC22A18) in lipid metabolism and in establishing the tumor phenotype of HepG2 cells.

Methods: SLC22A18-knockdown HepG2 cells were established by stable transfection with shRNA. Protein expression levels were measured by quantitative proteomics and Western blot analysis.