Membrane insertion mechanism of the caveola coat protein Cavin1.

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Altered brain network centrality in patients with retinal vein occlusion: a resting-state fMRI study.

AIM: To explore the intrinsic brain activity variations in retinal vein occlusion (RVO) subjects by using the voxel-wise degree centrality (DC) technique. METHODS: Twenty-one subjects with RVO and twenty-one healthy controls (HCs) were enlisted and underwent the resting-state functional magnetic resonance imaging (rs-fMRI) examination. The spontaneous cerebrum activity variations were inspected using the DC technology. The receiver operating characteristic (ROC) curve was implemented to distinguish the DC values of RVOs from HCs. The relationships between DC signal of definite regions of interest and the clinical characteristics in RVO group were evaluated by Pearson's correlation analysis. RESULTS: RVOs showed notably higher DC signals in right superior parietal lobule, middle frontal gyrus and left precuneus, but decreased DC signals in left middle temporal gyrus and bilateral anterior cingulated (BAC) when comparing with HCs. The mean DC value of RVOs in the BAC were negatively correlated with the anxiety and depression scale. CONCLUSION: RVO is associated aberrant intrinsic brain activity patterns in several brain areas including pain-related as well as visual-related regions, which might assist to reveal the latent neural mechanisms.

LRPPRC regulates metastasis and glycolysis by modulating autophagy and the ROS/HIF1-α pathway in retinoblastoma.

Retinoblastoma (RB) is the most common intraocular tumor among children. Leucine-rich pentatricopeptide repeat (PPR)-motif-containing protein (LRPPRC), a suppressor gene of autophagy, has been proven to play a regulatory role in tumor progression. However, little is known about functional roles and mechanisms of LRPPRC in RB progression. First, we performed a detailed analysis for RB and normal control. The expression of LRPPRC in the RB tissues was significantly higher than that in normal tissues. Moreover, LRPPRC suppression could repress tumor cell migration, invasion, glycolysis, and reactive oxygen species (ROS)/hypoxia-inducible factor-1α (HIF1-α) pathway activation by mediating autophagy. Furthermore, overexpression of HIF1-α partially reversed the above changes induced by LRPPRC knockdown. The regulation of LRPPRC on tumor metastasis and glycolysis was also validated by a xenograft tumor assay. In summary, LRPPRC could regulate metastasis and glycolysis of RB by mediating autophagy suppression and further activating the ROS/HIF1-α pathway, and LRPPRC could be a promising prognostic biomarker for RB.

Circular RNA COL1A2 promotes angiogenesis via regulating miR-29b/VEGF axis in diabetic retinopathy.

AIMS: The dysregulation of circular RNAs (circRNAs) has been implicated in the progression of diabetic retinopathy (DR). This study aims to explore the role and underlying mechanism of hsa_circ_0081108 (circCOL1A2) in DR. MATERIALS AND METHODS: circCOL1A2, vascular endothelial growth factor (VEGF) and miR-29b expression levels in human retinal microvascular endothelial cells (hRMECs) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting. The biological functions of hRMECs were evaluated by MTT, transwell, tube formation, and vascular permeability assays, respectively. The interaction between miR-29b and circCOL1A2/VEGF was determined by dual luciferase assay. The release of VEGF was examined by ELISA. The in vivo role of circCOL1A2 was further verified in streptozotocin (STZ)-induced DR in mice. The pathological changes and VEGF expression in retinal tissues were detected by hematoxylin and eosin (HE) and immunohistochemical staining. KEY FINDINGS: High glucose (HG) challenge led to increased circCOL1A2, VEGF, MMP-2, MMP-9 levels, but decreased miR-29b level in hRMECs. In addition, circCOL1A2 sponged miR-29b to promote VEGF expression. Silencing of circCOL1A2 inhibited HG-induced proliferation, migration, angiogenesis and vascular permeability of hRMECs via enhancing miR-29b expression. Moreover, circCOL1A2/miR-29b axis participated in HG-induced increase in angiogenesis-related protein expression. Finally, circCOL1A2 knockdown suppressed angiogenesis via regulating miR-29b/VEGF axis in DR mice. SIGNIFICANCE: circCOL1A2 facilities angiogenesis during the pathological progression of DR via regulating miR-29b/VEGF axis, suggesting that targeting circCOL1A2 may be a potential treatment for DR.

Abnormalities of interhemispheric functional connectivity in individuals with acute eye pain: a resting-state fMRI study.

AIM: To study the changes of the resting state functional connectivity (rsFC) between acute eye pain (EP) subjects and healthy controls (HCs) in the two hemispheres by using voxel-mirrored homotopic connectivity (VMHC) method. METHODS: Totally 20 patients with EP and 20 HCs were enrolled, sex, age, and education were matched, and all subjects were examined by functional magnetic resonance imaging (fMRI) scans at resting-state. The changes of rsFC between the hemispheres were evaluated by the VMHC method according to Gaussian random field (GRF) theory. In order to identify the VMHC, as biomarkers for distinguishing EP and from HC, the receiver operating characteristic curves (ROC) had been analyzed. The relationships were evaluated with Pearson correlation analysis between the mean VMHC signal values and clinical features in these patients. RESULTS: By comparing with health subjects, the significant decreased VMHC values was observed in lingual/calcarine (Brodmann area, BA 30), precentral/postcentral gyrus (PreCG/PosCG; BA 4) and medial frontal gyrus (MFG; BA 8) (false discovery rate corrected <0.01) in the acute EP individuals. The accuracy of area under curve was excellent indicated by the ROC curve analysis of each brain regions. CONCLUSION: Our study demonstrates preliminary evidence of disrupted interhemispheric rsFC in acute EP in sensorimotor and limbic system and somatosensory cortex, which might give some useful information for understanding the neurological mechanisms in acute EP individuals.

Altered Spontaneous Brain Activity in Patients with Classical Trigeminal Neuralgia Using Regional Homogeneity: A Resting-State Functional MRI Study.

OBJECTIVE: Neuroimaging studies have shown that patients with pain-related conditions have altered neuronal activity and structural functions. The purpose of this study was to investigate whether patients with classical trigeminal neuralgia (CTN) exhibit changes in corresponding neuronal activity via analysis of neuronal activity regional homogeneity (ReHo). METHODS: A total of 28 patients presenting with sore eyes (12 men and 16 women) were matched with 28 healthy controls (12 men and 16 women). All participants underwent functional magnetic resonance imaging (fMRI). This ReHo method was used to assess the consistency of changes in neural activity in various brain regions. The receiver operating characteristic (ROC) curve was applied to differentiate ReHo values of patients with CTN from ReHo values of healthy controls. Pearson's correlation analysis was applied to evaluate the correlation between ReHo values of different brain regions of patients with CTN and clinical manifestations. RESULTS: Compared with healthy controls, patients with CTN were found to have increased ReHo values in the inferior cerebellum bilaterally, right inferior temporal gyrus, right middle occipital gyrus, right fusiform gyrus, right superior frontal gyrus, and right precentral gyrus. ROC curve analysis of each brain region revealed near-perfect accuracy regarding the area under the curve. However, no correlation between ReHo values and clinical manifestations in patients with CTN was found. CONCLUSIONS: CTN is associated with altered neuronal networks in different areas of the brain. ReHo values all possess different degrees of change, implying that CTN has a certain impact on cerebral function.

Functional Connectivity of Paired Default Mode Network Subregions in Retinal Detachment.

PURPOSE: To explore the difference of the default mode network (DMN) in patients with retinal detachment (RD) by the study of the resting state functional connectivity (rs-FC). METHODS: A total of 30 patients with RD (16 men, 14 women) and 30 similarly matched normal controls (NCs) were examined and recorded with rs-fMRI. The DMN was divided into eight core regions, and each rs-FC map of each subregion was obtained. The receiver operating characteristic (ROC) curve was performed to classify the mean FC values of RD patients from NCs, and the interrelationships between the FC and each region were evaluated with Pearson's correlation analysis. RESULTS: Compared with NCs, there were significantly increased FC in the left medial temporal lobe (MTL.L) and posterior cingulate cortex (PCC), MTL.L and left hippocampus formation (HF.L), MTL.L and HF.R, MTL.L and left inferior parietal cortices (IPC.L), MTL.L and IPC.R in the RD group (P < 0.05). Nevertheless, no correlation between the FC values of each paired region and the manifestations was found in the RD group. ROC curve analysis showed that the accuracy of the area under the curve was excellent in MTL.L-HF.R and MTL.L-IPC.R and less reliable in MTL.L-PCC, MTL.L-HF.L, and MTL.L-IPC.L. CONCLUSIONS: The visual function impairments of RD patients were closely related to the DMN functional connections, which provided insight into the neural variation in RD patients and assisted in revealing the potential mechanisms of RD. TRANSLATIONAL RELEVANCE: This study provided insight into the neural variation in RD patients and assisted in revealing the potential mechanisms of RD.

Liver microsomal lipid enhances the activity and redox coupling of colocalized cytochrome P450 reductase-cytochrome P450 3A4 in nanodiscs.

The haem-containing mono-oxygenase cytochrome P450 3A4 (CYP3A4) and its redox partner NADPH-dependent cytochrome P450 oxidoreductase (CPR) are among the most important enzymes in human liver for metabolizing drugs and xenobiotic compounds. They are membrane-bound in the endoplasmic reticulum (ER). How ER colocalization and the complex ER phospholipid composition influence enzyme activity are not well understood. CPR and CYP3A4 were incorporated into phospholipid bilayer nanodiscs, both singly, and together in a 1 : 1 ratio, to investigate the significance of membrane insertion and the influence of varying membrane composition on steady-state reaction kinetics. Reaction kinetics were analysed using a fluorimetric assay with 7-benzyloxyquinoline as substrate for CYP3A4. Full activity of the mono-oxygenase system, with electron transfer from NADPH via CPR, could only be reconstituted when CPR and CYP3A4 were colocalized within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated separately into nanodiscs then mixed together, or when soluble forms of CPR were mixed with preassembled CYP3A4-nanodiscs. Membrane integration and colocalization are therefore essential for electron transfer. Liver microsomal lipid had an enhancing effect compared with phosphatidylcholine on the activity of CPR alone in nanodiscs, and a greater enhancing effect on the activity of CPR-CYP3A4 nanodisc complexes, which was not matched by a phospholipid mixture designed to mimic the ER composition. Furthermore, liver lipid enhanced redox coupling within the system. Thus, natural ER lipids possess properties or include components important for enhanced catalysis by CPR-CYP3A4 nanodisc complexes. Our findings demonstrate the importance of using natural lipid preparations for the detailed analysis of membrane protein activity.

Enzyme kinetics, inhibitors, mutagenesis and electron paramagnetic resonance analysis of dual-affinity nitrate reductase in unicellular N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801.

The assimilatory nitrate reductase (NarB) of N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801 is a monomeric enzyme with dual affinity for substrate nitrate. We purified the recombinant NarB of Cyanothece sp. PCC 8801 and further investigated it by enzyme kinetics analysis, site-directed mutagenesis, inhibitor kinetics analysis, and electron paramagnetic resonance (EPR) spectroscopy. The NarB showed 2 kinetic regimes at pH 10.5 or 8 and electron-donor conditions methyl viologen or ferredoxin (Fd). Fd-dependent NR assay revealed NarB with very high affinity for nitrate (K(m)1, ∼1µM; K(m)2, âˆ¼270µM). Metal analysis and EPR results showed that NarB contains a Mo cofactor and a [4Fe-4S] cluster. In addition, the R352A mutation on the proposed nitrate-binding site of NarB greatly altered both high- and low-affinity kinetic components. Furthermore, the effect of azide on the NarB of Cyanothece sp. PCC 8801 was more complex than that on the NarB of Synechococcus sp. PCC 7942 with its single kinetic regime. With 1mM azide, the kinetics of the wild-type NarB was transformed from 2 kinetic regimes to hyperbolic kinetics, and its activity was enhanced significantly under medium nitrate concentrations. Moreover, EPR results also suggested a structural difference between the two NarBs. Taken together, our results show that the NarB of Cyanothece sp. PCC 8801 contains only a single Mo-catalytic center, and we rule out that the enzyme has 2 independent, distinct catalytic sites. In addition, the NarB of Cyanothece sp. PCC 8801 may have a regulatory nitrate-binding site.

A novel six-rhodopsin system in a single archaeon.

Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered six-rhodopsin system in a single archaeon, Haloarcula marismortui, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in H. marismortui, and these were then overexpressed in Escherichia coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in H. marismortui.