Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer treatment because of their ...enhanced expression in various tumors. Here, we report five cryo-EM structures, at resolutions of 3.0–3.3 Å, of human MCT1 bound to lactate or inhibitors in the presence of Basigin-2, a single transmembrane segment (TM)-containing chaperon. MCT1 exhibits similar outward-open conformations when complexed with lactate or the inhibitors BAY-8002 and AZD3965. In the presence of the inhibitor 7ACC2 or with the neutralization of the proton-coupling residue Asp309 by Asn, similar inward-open structures were captured. Complemented by structural-guided biochemical analyses, our studies reveal the substrate binding and transport mechanism of MCTs, elucidate the mode of action of three anti-cancer drug candidates, and identify the determinants for subtype-specific sensitivities to AZD3965 by MCT1 and MCT4. These findings lay out an important framework for structure-guided drug discovery targeting MCTs.
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•A data processing strategy for cryo-analysis of small size membrane proteins•Structures of MCT1/Basigin-2 in distinct states at 3.0–3.3 Å resolutions•Substrate recognition and proton-coupled transport mechanism were elucidated•The mode of action of three anti-cancer drug candidates was unveiled
Structures of MCT1, a small highly flexible SLC-family transporter, in inward-open and outward open conformations, provide a window on how anti-cancer leads influence transport.
GLUT4 is the primary glucose transporter in adipose and skeletal muscle tissues. Its cellular trafficking is regulated by insulin signaling. Failed or reduced plasma membrane localization of GLUT4 is ...associated with diabetes. Here, we report the cryo-EM structures of human GLUT4 bound to a small molecule inhibitor cytochalasin B (CCB) at resolutions of 3.3 Å in both detergent micelles and lipid nanodiscs. CCB-bound GLUT4 exhibits an inward-open conformation. Despite the nearly identical conformation of the transmembrane domain to GLUT1, the cryo-EM structure reveals an extracellular glycosylation site and an intracellular helix that is invisible in the crystal structure of GLUT1. The structural study presented here lays the foundation for further mechanistic investigation of the modulation of GLUT4 trafficking. Our methods for cryo-EM analysis of GLUT4 will also facilitate structural determination of many other small size solute carriers.
Human glucose transporters (GLUTs) are responsible for cellular uptake of hexoses. Elevated expression of GLUTs, particularly GLUT1 and GLUT3, is required to fuel the hyperproliferation of cancer ...cells, making GLUT inhibitors potential anticancer therapeutics. Meanwhile, GLUT inhibitor-conjugated insulin is being explored to mitigate the hypoglycemia side effect of insulin therapy in type 1 diabetes. Reasoning that exofacial inhibitors of GLUT1/3 may be favored for therapeutic applications, we report here the engineering of a GLUT3 variant, designated GLUT3exo, that can be probed for screening and validating exofacial inhibitors. We identify an exofacial GLUT3 inhibitor SA47 and elucidate its mode of action by a 2.3 Å resolution crystal structure of SA47-bound GLUT3. Our studies serve as a framework for the discovery of GLUTs exofacial inhibitors for therapeutic development.
This study aims addresses the challenge of gesture recognition in smart sports stadiums through the fusion enhancement of YOLOv5 and Copula Bayesian Classifier algorithm. By leveraging FasterNet for ...YOLOv5 transformation, integrating the Convolutional Block Attention Module (CBAM) as an attention mechanism, and merging it with the Copula Bayesian Classifier, we propose a tandem model for robust gesture recognition in the complex environments of sports venues. Extensive training on a substantial dataset yields an efficient and accurate gesture recognition model. Our findings showcase the effectiveness of the proposed algorithm, achieving a remarkable accuracy rate of 99.2% in identifying gesture categories swiftly. This advancement holds significant implications for future human-computer interaction in smart sports stadiums, offering a more seamless and intelligent mode of interaction for both spectators and athletes. Ultimately, it enhances user experience and management efficiency within smart sports venues.
Cigarette smoking (CS) is a strong risk factor for idiopathic pulmonary fibrosis (IPF). It can activate lung fibroblasts (LF) by inducing redox imbalance. We previously showed that clearing ...mitochondrial reactive oxygen species (mtROS) protects against CS-induced pulmonary fibrosis. However, the precise mechanisms of mtROS in LF need further investigation. Here we focused on mtROS to elucidate how it was regulated by CS in LF and how it contributed to LF activation.
We treated cells with 1% cigarette smoking extract (CSE) and examined mtROS level by MitoSOX
indicator. And the effect of CSE on expression of SIRT1, SOD2, mitochondrial NOX4 (mtNOX4), fatty acid oxidation (FAO)-related protein PPARα and CPT1a and LF activation marker Collagen I and α-SMA were detected. Nile Red staining was performed to show cellular lipid content. Then, lipid droplets, autophagosome and lysosome were marked by Bodipy 493/503, LC3 and LAMP1, respectively. And lipophagy was evaluated by the colocalization of lipid droplets with LC3 and LAMP1. The role of autophagy on lipid metabolism and LF activation were explored. Additionally, the effect of mitochondria-targeted ROS scavenger mitoquinone and SIRT1 activator SRT1720 on mitochondrial oxidative stress, autophagy flux, lipid metabolism and LF activation were investigated in vitro and in vivo.
We found that CS promoted mtROS production by increasing mtNOX4 and decreasing SOD2. Next, we proved mtROS inhibited the expression of PPARα and CPT1a. It also reduced lipophagy and upregulated cellular lipid content, suggesting lipid metabolism was disturbed by CS. In addition, we showed both insufficient FAO and lipophagy resulted from blocked autophagy flux caused by mtROS. Moreover, we uncovered decreased SIRT1 was responsible for mitochondrial redox imbalance. Furthermore, we proved that both SRT1720 and mitoquinone counteracted the effect of CS on NOX4, SOD2, PPARα and CPT1a in vivo.
We demonstrated that CS decreased SIRT1 to activate LF through dysregulating lipid metabolism, which was due to increased mtROS and impaired autophagy flux. These events may serve as therapeutic targets for IPF patients.
GLUT1 facilitates the down-gradient translocation of D-glucose across cell membrane in mammals. XylE, an Escherichia coli homolog of GLUT1, utilizes proton gradient as an energy source to drive ...uphill D-xylose transport. Previous studies of XylE and GLUT1 suggest that the variation between an acidic residue (Asp27 in XylE) and a neutral one (Asn29 in GLUT1) is a key element for their mechanistic divergence. In this work, we combined computational and biochemical approaches to investigate the mechanism of proton coupling by XylE and the functional divergence between GLUT1 and XylE. Using molecular dynamics simulations, we evaluated the free energy profiles of the transition between inward- and outward-facing conformations for the apo proteins. Our results revealed the correlation between the protonation state and conformational preference in XylE, which is supported by the crystal structures. In addition, our simulations suggested a thermodynamic difference between XylE and GLUT1 that cannot be explained by the single residue variation at the protonation site. To understand the molecular basis, we applied Bayesian network models to analyze the alteration in the architecture of the hydrogen bond networks during conformational transition. The models and subsequent experimental validation suggest that multiple residue substitutions are required to produce the thermodynamic and functional distinction between XylE and GLUT1. Despite the lack of simulation studies with substrates, these computational and biochemical characterizations provide unprecedented insight into the mechanistic difference between proton symporters and uniporters.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Heart rate variability (HRV) reflects the regulating effect of autonomic nerve on cardiovascular functions and is strongly influenced by respiratory rhythm. For more convenient monitoring ...physiological or pathological information about heart and autonomic nerves during sleep than conventional systems, a smart mattress that could measure electrocardiogram (ECG) and respiratory signals through clothes was designed. The system mainly consists of three electrodes that are made of flexible silver fiber conductive fabric, a signal preprocessing module and a user interface. To reduce the effect of surrounding electromagnetic radiation on the electrodes, two equipotential shielding layers are placed under the two active electrodes, respectively. To verify the proposed system, experiments in four sleeping postures (supine, left lateral, right lateral, and prone) were carried out. The results showed that the proposed system could successfully measure ECG signals and extract respiratory signals from them in all sleeping postures. Finally, to study respiratory sinus arrhythmia during sleep, an experiment was conducted to measure ECG and respiratory signals of 7 subjects under unconstrained sleep over night. Then, root mean square of successive differences (RMSSD) and peak-valley respiratory sinus arrhythmia (pvRSA) that can reflect the vagus nerve tension were calculated. The results showed that all subjects had varying degrees of respiratory sinus arrhythmia, with the mean and standard deviation of <inline-formula> <tex-math notation="LaTeX">176 \pm 80\textit {ms} </tex-math></inline-formula> and <inline-formula> <tex-math notation="LaTeX">288 \pm 126\textit {ms} </tex-math></inline-formula>, for the RMSSD and the pvRSA respectively. The RMSSD and the pvRSA of the subject with most obvious arrhythmia were <inline-formula> <tex-math notation="LaTeX">346\textit {ms} </tex-math></inline-formula> and <inline-formula> <tex-math notation="LaTeX">552\textit {ms} </tex-math></inline-formula> respectively. The results also indicate the potential application of the proposed system in detecting sleep diseases such as sleep apnea.
The present study explores a three-dimensional deformation monitoring method for the better delineation of the surface subsidence range in coal mining by combining the mining subsidence law with the ...geometries of SAR imaging. The mining surface subsidence of the filling working face in Shandong, China, from March 2018 to June 2021, was obtained with 97 elements of Sentinel-1A data, the small baseline subset (SBAS) technique, and the proposed method, respectively. By comparison with the ground leveling of 46 observation stations, it is shown that the average standard deviation of the SBAS monitoring results is 10.3 mm; with this deviation, it is difficult to satisfy the requirements for the delimitation of the mining impact area. Meanwhile, the average standard deviation of the vertical deformation obtained by the proposed method is 6.2 mm. Compared to the SBAS monitoring accuracy, the monitoring accuracy of the proposed method is increased by 39.8%; thus, it meets the requirements for the precise delineation of the surface subsidence range for backfill mining.
Idiopathic pulmonary fibrosis is a chronic progressive, lethal disease in which ectopic lung fibroblast (LF) activation plays a vital part. We have previously shown that alamandine (ALA) exerts ...anti-fibrosis effects via the MAS-related G-protein coupled receptor D (MrgD). Here, we further investigate how it moderates transforming growth factor β1 (TGF-β1)-induced LF activation by regulating glucose metabolism and mitochondria autophagy (mitophagy).
In vitro, we examined glycolysis-related protein hexokinase 2 (HK2), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), and lactic acid in cells treated with TGF-β1. The oxygen consumption rate and the extracellular acidification rate were detected using Seahorse assays. Then, mitophagy was evaluated using transmission electron microscopy, mt-Keima, and the co-localization of Parkin and COX IV with LC3 and LAMP1, respectively. The autophagic degradation of HK2 and PFKFB3 was detected by 3MA and bafilomycin A1 and assessed by their co-localization with LC3 and LAMP1, respectively. The effects of ALA on LF activation markers collagen I and α-SMA were detected. The effects of ALA on glucose metabolism, mitophagy, and the activation of LF were also investigated in vivo.
We found that the ALA/MrgD axis improved TGF-β1-mediated LF activation by repressing glycolysis by downregulating HK2 and PFKFB3 expression. Lactic acid sustained positive feedback between glycolysis and LF activation by maintaining the expression of HK2 and PFKFB3. We also showed that glycolysis enhancement resulted from blocking the autophagic degradation of HK2 and PFKFB3 while upregulated mRNA levels by TGF-β1, while all of those improved by ALA adding. Importantly, we determined that moderation of Parkin/LC3-mediated mitophagy by TGF-β1 also promotes glycolysis but is reversed by ALA. Furthermore, we proved that ALA counteracts the effects of bleomycin on HK2, PFKFB3, LC3, Parkin, and LF activation in vivo.
In this study, we show that the ALA/MrgD axis prevents TGF-β1-mediated fibroblast activation via regulation of aerobic glycolysis and mitophagy.
Acting as a brain stimulant, coffee resulted in heightening alertness, keeping arousal, improving executive speed, maintaining vigilance, and promoting memory, which are associated with attention, ...mood, and cognitive function. Functional near-infrared spectroscopy (fNIRS) is a noninvasive optical method to monitor brain activity by measuring the absorption of the near-infrared light through the intact skull. This study is aimed at acquiring brain activation during executing task performance. The aim is to explore the effect of coffee on cognitive function by the fNIRS neuroimaging method, particularly on the prefrontal cortex regions. The behavioral experimental results on 31 healthy subjects with a Stroop task indicate that coffee can easily and effectively modulate the execute task performance by feedback information of the response time and accuracy rate. The findings of fNIRS showed that apparent hemodynamic changes were detected in the bilateral VLPFC regions and the brain activation regions varied with different coffee conditions.
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