Gene-Specific Control of tRNA Expression by RNA Polymerase II.

Increasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.

Role of cellular retinol-binding protein, type 1 and retinoid homeostasis in the adult mouse heart: A multi-omic approach.

The main active metabolite of Vitamin A, all-trans retinoic acid (RA), is required for proper cellular function and tissue organization. Heart development has a well-defined requirement for RA, but there is limited research on the role of RA in the adult heart. Homeostasis of RA includes regulation of membrane receptors, chaperones, enzymes, and nuclear receptors. Cellular retinol-binding protein, type 1 (CRBP1), encoded by retinol-binding protein, type 1 (Rbp1), regulates RA homeostasis by delivering vitamin A to enzymes for RA synthesis and protecting it from non-specific oxidation. In this work, a multi-omics approach was used to characterize the effect of CRBP1 loss using the Rbp1-/- mouse. Retinoid homeostasis was disrupted in Rbp1-/- mouse heart tissue, as seen by a 33% and 24% decrease in RA levels in the left and right ventricles, respectively, compared to wild-type mice (WT). To further inform on the effect of disrupted RA homeostasis, we conducted high-throughput targeted metabolomics. A total of 222 metabolite and metabolite combinations were analyzed, with 33 having differential abundance between Rbp1-/- and WT hearts. Additionally, we performed global proteome profiling to further characterize the impact of CRBP1 loss in adult mouse hearts. More than 2606 unique proteins were identified, with 340 proteins having differential expression between Rbp1-/- and WT hearts. Pathway analysis performed on metabolomic and proteomic data revealed pathways related to cellular metabolism and cardiac metabolism were the most disrupted in Rbp1-/- mice. Together, these studies characterize the effect of CRBP1 loss and reduced RA in the adult heart.

Cellular retinol-binding protein 1: a therapeutic and diagnostic tumor marker.

Cellular Retinol Binding Protein 1 (CRBP1) gene is a protein coding gene located on human chromosome 3q21, which codifies a protein named CRBP1. CRBP1 is widely expressed in many tissues as a chaperone protein to regulate the uptake, subsequent esterification and bioavailability of retinol. CRBP1 combines retinol and retinaldehyde with high affinity to protect retinoids from non-specific oxidation, and transports retinoids to specific enzymes to promote the biosynthesis of retinoic acid. The vital role of CRBP1 in retinoids metabolism has been gradually discovered, which has been implicated in tumorigenesis. However, the precise functions of CRBP1 in different diseases are still poorly understood. The purpose of this review is to provide an overview of the role of CRBP1 in various diseases, especially in both the promotion and inhibition of cancers, which may also offer a novel biomarker and potential therapeutic target for human diseases.

Retinol-binding protein 2 (RBP2): biology and pathobiology.

Retinol-binding protein 2 (RBP2; originally cellular retinol-binding protein, type II (CRBPII)) is a 16 kDa cytosolic protein that in the adult is localized predominantly to absorptive cells of the proximal small intestine. It is well established that RBP2 plays a central role in facilitating uptake of dietary retinoid, retinoid metabolism in enterocytes, and retinoid actions locally within the intestine. Studies of mice lacking Rbp2 establish that Rbp2 is not required in times of dietary retinoid-sufficiency. However, in times of dietary retinoid-insufficiency, the complete lack of Rbp2 gives rise to perinatal lethality owing to RBP2 absence in both placental (maternal) and neonatal tissues. Moreover, when maintained on a high-fat diet, Rbp2-knockout mice develop obesity, glucose intolerance and a fatty liver. Unexpectedly, recent investigations have demonstrated that RBP2 binds long-chain 2-monoacylglycerols (2-MAGs), including the canonical endocannabinoid 2-arachidonoylglycerol, with very high affinity, equivalent to that of retinol binding. Crystallographic studies establish that 2-MAGs bind to a site within RBP2 that fully overlaps with the retinol binding site. When challenged orally with fat, mucosal levels of 2-MAGs in Rbp2 null mice are significantly greater than those of matched controls establishing that RBP2 is a physiologically relevant MAG-binding protein. The rise in MAG levels is accompanied by elevations in circulating levels of the hormone glucose-dependent insulinotropic polypeptide (GIP). It is not understood how retinoid and/or MAG binding to RBP2 affects the functions of this protein, nor is it presently understood how these contribute to the metabolic and hormonal phenotypes observed for Rbp2-deficient mice.

Retinol binding protein 1-dependent activation of NF- κB signaling enhances the malignancy of non-glioblastomatous diffuse gliomas.

Nonglioblastomatous diffuse glioma (non-GDG) is a heterogeneous neuroepithelial tumor that exhibits a varied survival range from 4 to 13 years based on the diverse subtypes. Recent studies demonstrated novel molecular markers can predict prognosis for non-GDG patients; however, these findings as well as pathological classification strategies show obvious limitations on malignant transition due to the heterogeneity among non-GDGs. Therefore, developing reliable prognostic biomarkers and therapeutic targets have become an urgent need for precisely distinguishing non-GDG subtypes, illuminating the underlying mechanism. Nuclear factor κß (NF-κB) has been proved to be a significant nuclear transcriptional regulator with specific DNA-binding sequences to participate in multiple pathophysiological processes. However, the underlying mechanism of NF-κB activation still needs to be further investigated. Herein, our results indicated retinol-binding protein 1 (RBP1) was significantly upregulated in the IDHWT and 1p19qNon co-del non-GDG subtypes and enriched RBP1 expression was markedly correlated with more severe outcomes. Additionally, malignant signatures of the non-GDG cells including proliferation, migration, invasion, and self-renewal were significantly suppressed by lentiviral knockdown of RBP1. To further explore the underlying molecular mechanism, bioinformatics analysis was performed using databases, and the results demonstrated RBP1 was strongly correlated with tumor necrosis factor α (TNFα)-NF-κB signaling. Moreover, exogenous silencing of RBP1 reduced phosphorylation of IkB-kinase α (IKKα) and thus decreased NF-κB expression via decreasing the degradation of the IκBα protein. Altogether, these data suggested RBP1-dependent activation of NF-κB signaling promoted malignancy of non-GDG, indicating that RBP1 could be a reliable prognostic biomarker and potential therapeutic target for non-GDG.

Retinol binding protein 1 affects Xenopus anterior neural development via all-trans retinoic acid signaling.

BACKGROUND: Retinol binding protein 1 (Rbp1) acts as an intracellular regulator of vitamin A metabolism and retinoid transport. In mice, Rbp1 deficiency decreases the capacity of hepatic stellate cells to take up all-trans retinol and sustain retinyl ester stores. Furthermore, Rbp1 is crucial for visual capacity. Although the function of Rbp1 has been studied in the mature eye, its role during early anterior neural development has not yet been investigated in detail. RESULTS: We showed that rbp1 is expressed in the eye, anterior neural crest cells (NCCs) and prosencephalon of the South African clawed frog Xenopus laevis. Rbp1 knockdown led to defects in eye formation, including microphthalmia and disorganized retinal lamination, and to disturbed induction and differentiation of the eye field, as shown by decreased rax and pax6 expression. Furthermore, it resulted in reduced rax expression in the prosencephalon and affected cranial cartilage. Rbp1 inhibition also interfered with neural crest induction and migration, as shown by twist and slug. Moreover, it led to a significant reduction of the all-trans retinoic acid target gene pitx2 in NCC-derived periocular mesenchyme. The Rbp1 knockdown phenotypes were rescued by pitx2 RNA co-injection. CONCLUSION: Rbp1 is crucial for the development of the anterior neural tissue.

[Effect of RBP2 gene silencing on proliferation, migration and invasion of ovarian epithelial cancer SKOV3/DDP cells and its mechanism].

Objective: To explore the effect of down-regulation of retinol binding protein 2 (RBP2) expression on the biological characteristics of ovarian cancer cells and its mechanism. Methods: Knockdown of RBP2 and cisplatin (DDP)-resistant ovarian cancer cell line SKOV3/DDP-RBP2i was established, the negative control group and blank control group were also set. Cell counting kit 8 (CCK-8) was used to detect the cell proliferation ability, flow cytometry was used to detect cell apoptosis, scratch test and Transwell invasion test were used to detect cell migration and invasion ability, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and western blot were used to detect the expressions of molecular markers related to epithelial-mesenchymal transition (EMT). The effect of RBP2 on the growth of ovarian cancer was verified through experiment of transplanted tumors in nude mice, and the relationships between RBP2 expression and tumor metastasis and patient prognosis were analyzed using the clinical data of ovarian cancer in TCGA database. Results: After down-regulating the expression of RBP2, the proliferation ability of SKOV3/DDP cell was significantly reduced. On the fifth day, the proliferation activities of SKOV3/DDP-RBP2i group, negative control group and blank control group were (56.67±4.16)%, (84.67±3.51) and (87.00±4.00)% respectively, with statistically significant difference (P<0.001). The apoptosis rate of SKOV3/DDP-RBP2i group was (14.19±1.50)%, higher than (8.77±0.75)% of the negative control group and (7.48±0.52)% of the blank control group (P<0.001). The number of invasive cells of SKOV3/DDP-RBP2i group was (55.20±2.39), lower than (82.60±5.18) and (80.80±7.26) of the negative control group and the blank control group, respectively (P<0.001). The scratch healing rate of SKOV3/DDP-RBP2i group was (28.47±2.72)%, lower than (50.58±4.06)% and (48.92±4.63)% of the negative control group and the blank control group, respectively (P<0.001). The mRNA and protein expressions of E-cadherin in the SKOV3/DDP-RBP2i group were higher than those in the negative control group (P=0.015, P<0.001) and the blank control group (P=0.006, P<0.001). The mRNA and protein expression of N-cadherin in SKOV3/DDP-RBP2i group were lower than those in the negative control group (P=0.012, P<0.001) and the blank control group (P=0.005, P<0.001). The mRNA and protein expressions of vimentin in SKOV3/DDP-RBP2i group were also lower than those in the negative control group (P=0.016, P=0.001) and the blank control group (P=0.011, P=0.001). Five weeks after the cells inoculated into the nude mice, the tumor volume of SKOV3/DDP-RBP2i group, negative control group and blank control group were statistically significant different. The tumor volume of SKOV3/DDP-RBP2i group was smaller than those of negative control group and blank control group (P=0.001). Bioinformatics analysis showed that the expression of RBP2 in patients with metastatic ovarian cancer was higher than that without metastasis (P=0.043), and the median overall survival of ovarian cancer patients with high RBP2 expression was 41 months, shorter than 69 months of low RBP2 expression patients (P<0.001). Conclusion: Downregulation of the expression of RBP2 in SKOV3/DDP cells can inhibit cell migration and invasion, and the mechanism may be related to the inhibition of EMT.

Analysis of exome-sequenced UK Biobank subjects implicates genes affecting risk of hyperlipidaemia.

Rare genetic variants in LDLR, APOB and PCSK9 are known causes of familial hypercholesterolaemia and it is expected that rare variants in other genes will also have effects on hyperlipidaemia risk although such genes remain to be identified. The UK Biobank consists of a sample of 500,000 volunteers and exome sequence data is available for 50,000 of them. 11,490 of these were classified as hyperlipidaemia cases on the basis of having a relevant diagnosis recorded and/or taking lipid-lowering medication while the remaining 38,463 were treated as controls. Variants in each gene were assigned weights according to rarity and predicted impact and overall weighted burden scores were compared between cases and controls, including population principal components as covariates. One biologically plausible gene, HUWE1, produced statistically significant evidence for association after correction for testing 22,028 genes with a signed log10 p value (SLP) of -6.15, suggesting a protective effect of variants in this gene. Other genes with uncorrected p < .001 are arguably also of interest, including LDLR (SLP = 3.67), RBP2 (SLP = 3.14), NPFFR1 (SLP = 3.02) and ACOT9 (SLP = -3.19). Gene set analysis indicated that rare variants in genes involved in metabolism and energy can influence hyperlipidaemia risk. Overall, the results provide some leads which might be followed up with functional studies and which could be tested in additional data sets as these become available. This research has been conducted using the UK Biobank Resource.

Identification of RBP4 from bighead carp (Hypophthalmichthys nobilis) / silver carp (Hypophthalmichthys molitrix) and effects of CpG ODN on RBP4 expression under A. hydrophila challenge.

Retinol-binding protein 4 (RBP4) is known as a highly conserved adipokine for immune activation. Aeromonas hydrophila (A. hydrophila) is the most common zoonotic pathogen in aquaculture, which causes serious economic losses to aquaculture, especially to bighead carp (Hypophthalmichthys nobilis, H. nobilis) and silver carp (Hypophthalmichthys molitrix, H. molitrix). Recent studies along with our previous findings have shown that synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) can play a good role in aquatic animals against infection. In order to clarify the relationship between CpG ODN and RBP4 under A. hydrophila infection, firstly, full-length RBP4 cDNAs from H. nobilis and H. molitrix were cloned. And characteristics of RBP4, including sequence and structure, tissue distribution and genetic evolution were analyzed. In addition, mRNA expression levels of RBP4, cytokine, toll-like receptors (TLRs), morbidity and survival rates of H. nobilis and H. molitrix were observed post CpG ODN immunization or following challenge. The results indicated that hn/hm_RBP4 (RBP4 genes obtained from H. nobilis and H. molitrix) had the highest homology with Megalobrama amblycephala. Distribution data showed that the expression level of hn_RBP4 mRNA was higher than that of hm_RBP4. After CpG ODN immunization followed by A.hydrophila challenge, significantly higher survival was observed in both carps, together with up-regulated RBP4 expression. Meanwhile, hn/hm_IL-1ß level was relatively flat (and decreased), hn/hm_IFN-γ, hn/hm_TLR4 and hn/hm_TLR9 levels increased significantly, but hn/hm_STRA6 showed no significant change, compared with control. Moreover, CpG ODN immunization could induce stronger immune protective responses (higher IFN-γ/gentle IL-1ß level and lower morbidity/higher survival rate) against A. hydrophila in H. nobilis, along with higher RBP4 level, when compared with that in H. molitrix. These results demonstrated that RBP4 was well involved in the immune protection of CpG ODN. Based on the results, we speculated that in the case of A. hydrophila infection, TLR9 signaling pathway was activated by CpG ODN. Subsequently, CpG ODN up-regulated RBP4, and RBP4 activated TLR4 signaling pathway. Then TLR4 and TLR9 synergistically improved the anti-infection responses. Our findings have good significance for improving resistance to pathogen infection in freshwater fish.

Cellular retinol binding protein 1 transfection reduces proliferation and AKT-related gene expression in H460 non-small lung cancer cells.

In recent years, new treatments with novel action mechanisms have been explored for advanced non-small cell lung cancer (NSCLC). Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. The purpose of this study was to investigate the effect of Cellular retinol binding protein-1 (CRBP-1) transfection in H460 human NSCLC cell line, normally not expressing CRBP-1. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP-1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by t-student test. CRBP-1+ showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBP-1+ H460 cells associated to the down-regulation of pAKT/pERK/pEGFR-related genes. In particular, gene array documented the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, p27, Jun. Restoration of CRBP-1 expression in H460 cells reduced proliferation and viability in both basal condition and after atRA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBP-1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.

Dynamic site-specific recruitment of RBP2 by pocket protein p130 modulates H3K4 methylation on E2F-responsive promoters.

The Histone 3 lysine 4 methylation (H3K4me3) mark closely correlates with active transcription. E2F-responsive promoters display dynamic changes in H3K4 methylation during the course of cell cycle progression. However, how and when these marks are reset, is not known. Here we show that the retinoblastoma binding protein RBP2/KDM5A, capable of removing tri-methylation marks on H3K4, associates with the E2F4 transcription factor via the pocket protein-p130-in a cell-cycle-stage specific manner. The association of RBP2 with p130 is LxCxE motif dependent. RNAi experiments reveal that p130 recruits RBP2 to E2F-responsive promoters in early G1 phase to bring about H3K4 demethylation and gene repression. A point mutation in LxCxE motif of RBP2 renders it incapable of p130-interaction and hence, repression of E2F-regulated gene promoters. We also examine how RBP2 may be recruited to non-E2F responsive promoters. Our studies provide insight into how the chromatin landscape needs to be adjusted rapidly and periodically during cell-cycle progression, concomitantly with temporal transcription, to bring about expression/repression of specific gene sets.

Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca2+ channels.

Fast neurotransmitter release from ribbon synapses via Ca2+-triggered exocytosis requires tight coupling of L-type Ca2+ channels to release-ready synaptic vesicles at the presynaptic active zone, which is localized at the base of the ribbon. Here, we used genetic, electrophysiological, and ultrastructural analyses to probe the architecture of ribbon synapses by perturbing the function of RIM-binding proteins (RBPs) as central active-zone scaffolding molecules. We found that genetic deletion of RBP1 and RBP2 did not impair synapse ultrastructure of ribbon-type synapses formed between rod bipolar cells (RBCs) and amacrine type-2 (AII) cells in the mouse retina but dramatically reduced the density of presynaptic Ca2+ channels, decreased and desynchronized evoked neurotransmitter release, and rendered evoked and spontaneous neurotransmitter release sensitive to the slow Ca2+ buffer EGTA. These findings suggest that RBPs tether L-type Ca2+ channels to the active zones of ribbon synapses, thereby synchronizing vesicle exocytosis and promoting high-fidelity information transfer in retinal circuits.

Engineering the hCRBPII Domain-Swapped Dimer into a New Class of Protein Switches.

Protein conformational switches or allosteric proteins play a key role in the regulation of many essential biological pathways. Nonetheless, the implementation of protein conformational switches in protein design applications has proven challenging, with only a few known examples that are not derivatives of naturally occurring allosteric systems. We have discovered that the domain-swapped (DS) dimer of hCRBPII undergoes a large and robust conformational change upon retinal binding, making it a potentially powerful template for the design of protein conformational switches. Atomic resolution structures of the apo- and holo-forms illuminate a simple, mechanical movement involving sterically driven torsion angle flipping of two residues that drive the motion. We further demonstrate that the conformational "readout" can be altered by addition of cross-domain disulfide bonds, also visualized at atomic resolution. Finally, as a proof of principle, we have created an allosteric metal binding site in the DS dimer, where ligand binding results in a reversible 5-fold loss of metal binding affinity. The high resolution structure of the metal-bound variant illustrates a well-formed metal binding site at the interface of the two domains of the DS dimer and confirms the design strategy for allosteric regulation.

Chromatin remodeling: demethylating H3K4me3 of type I IFNs gene by Rbp2 through interacting with Piasy for transcriptional attenuation.

Type I IFNs (IFNIs) are involved in the course of antiviral and antimicrobial activities; however, robust inductions of these can lead to host immunopathology. We have reported that the Pias (protein inhibitor of activated signal transducer and activator of transcription) family member, Piasy, possesses the ability to suppress IFNI transcriptions in mouse embryonic fibroblasts (MEFs), yet the specific molecular mechanism by which it acts remains elusive. Here, we identify that the H3K4me3 levels, one activation mark of genes, in MEFs that were stimulated by poly(I:C) were impaired by Piasy in the IFN-ß gene. Piasy bound to the promoter region of the IFN-ß gene in MEFs. Meanwhile, retinoblastoma binding protein 2 (Rbp2) was proven to be the only known and novel H3K4me3 demethylase that interacted with Piasy. Overexpression of Rbp2, but not its enzymatically inactive mutant Rbp2H483G/E485Q, retarded the transcription activities of IFNI, whereas small interfering RNA-mediated or short hairpin RNA-mediated knockdown of Rbp2 enhanced IFNI promoter responses. Above all, coexpression of Piasy and Rbp2 led to statistically less IFNI induction than overexpression of either Piasy or Rbp2 alone. Mechanistically, Piasy bound to the Jmjc domain (451-503 aa) of Rbp2 via its PINIT domain (101-218 aa), which is consistent with the domain required for their attenuation of transcription and H3K4me3 levels of IFNI genes. Our study demonstrates that Piasy may prevent exaggerated transcription of IFNI by Rbp2-mediated demethylation of H3K4me3 of IFNI, avoiding excessive immune responses.-Yu, X., Chen, H., Zuo, C., Jin, X., Yin, Y., Wang, H., Jin, M., Ozato, K., Xu, S. Chromatin remodeling: demethylating H3K4me3 of type I IFNs gene by Rbp2 through interacting with Piasy for transcriptional attenuation.

A Genetically Encoded Ratiometric pH Probe: Wavelength Regulation-Inspired Design of pH Indicators.

Mutants of human cellular retinol-binding protein II (hCRBPII) were engineered to bind a julolidine retinal analogue for the purpose of developing a ratiometric pH sensor. The design relied on the electrostatic influence of a titratable amino acid side chain, which affects the absorption and, thus, the emission of the protein/fluorophore complex. The ratio of emissions obtained at two excitation wavelengths that correspond to the absorption of the two forms of the protein/fluorophore complex, leads to a concentration-independent measure of pH.

9-cis-13,14-Dihydroretinoic Acid Is an Endogenous Retinoid Acting as RXR Ligand in Mice.

The retinoid X receptors (RXRs) are ligand-activated transcription factors which heterodimerize with a number of nuclear hormone receptors, thereby controlling a variety of (patho)-physiological processes. Although synthetic RXR ligands are developed for the treatment of various diseases, endogenous ligand(s) for these receptors have not been conclusively identified. We show here that mice lacking cellular retinol binding protein (Rbp1-/-) display memory deficits reflecting compromised RXR signaling. Using HPLC-MS and chemical synthesis we identified in Rbp1-/- mice reduced levels of 9-cis-13,14-dihydroretinoic acid (9CDHRA), which acts as an RXR ligand since it binds and transactivates RXR in various assays. 9CDHRA rescues the Rbp1-/- phenotype similarly to a synthetic RXR ligand and displays similar transcriptional activity in cultured human dendritic cells. High endogenous levels of 9CDHRA in mice indicate physiological relevance of these data and that 9CDHRA acts as an endogenous RXR ligand.

Comprehensive molecular exploration identified promoter DNA methylation of the CRBP1 gene as a determinant of radiation sensitivity in rectal cancer.

BACKGROUND: Neoadjuvant chemoradiotherapy (NCRT) for advanced rectal cancer (RC) is a well-evidenced therapy; however, some RC patients have no therapeutic response. Patient selection for NCRT so that non-responsive patients are excluded has been subjective. To date, no molecular markers indicating radiation sensitivity have been reported. METHODS: We irradiated six colorectal cancer (CRC) cell lines and identified HCT116 cells as radiation-sensitive and HCT15 and DLD-1 cells as radiation resistant. Using a microarray, we selected candidate radiation sensitivity marker genes by choosing genes whose expression was consistent with a radiation-resistant or sensitive cell phenotype. RESULTS: Among candidate genes, cellular retinol binding protein 1 (CRBP1) was of particular interest because it was not only induced in HCT116 cells by tentative 10 Gy radiation treatments, but also its expression was increased in HCT116-derived radiation-resistant cells vs parental cells. Forced expression of CRBP1 decreased the viability of both HCT15 and DLD-1 cells in response to radiation therapy. We also confirmed that CRBP1 was epigenetically silenced by hypermethylation of its promoter DNA, and that the quantitative methylation value of CRBP1 significantly correlated with histological response in RC patients with NCRT (P=0.031). CONCLUSIONS: Our study identified CRBP1 as a radiation-sensitive predictor in RC.

Functions of Intracellular Retinoid Binding-Proteins.

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

Proteomic changes in the liver of Channa striatus in response to high temperature stress.

The present study was undertaken to investigate the proteomic changes in the liver of murrel Channa striatus exposed to high temperature stress. Fishes were exposed to 36°C for 4 days and liver proteome changes were analyzed using gel- based proteomics, i.e. 2DE, MALDI-TOF/TOF-MS, and validation by transcript analysis. The study showed, besides others, increased abundance of two sets of proteins, the antioxidative enzymes superoxide dismutase (SOD), ferritin, cellular retinol binding protein (CRBP), glutathione-S-transferase (GST), and the chaperones HSP60 and protein disulfide isomerase; this was validated by transcript analysis. The proteome data are available via ProteomeXchange with identifier PXD002608. Further, gene expression analysis was also carried out in the fishes exposed to thermal stress for longer durations (30 days experimental exposure in laboratory and for 30 days beyond, taking Channa collected from a hot spring runoff at 36-38°C); sod, gst, crbp, and hsp60 were found to continue to remain upregulated at eight-, 2.5-, 2.4-, and 2.45-fold, respectively, in the hot spring runoff fish. Pathway analysis showed that the upregulations of the antioxidant enzymes as well as molecular chaperones are induced by the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Thus, while short-term heat stress tolerance involves the antioxidative enzymes SOD, ferritin, CRBP, GST, and chaperones HSP60 and protein disulfide isomerase, adaptation under chronic heat stress is associated with SOD, CRBP, GST, and HSP60.

Tuning the Protein-Induced Absorption Shifts of Retinal in Engineered Rhodopsin Mimics.

Rational design of light-capturing properties requires understanding the molecular and electronic structure of chromophores in their native chemical or biological environment. We employ here large-scale quantum chemical calculations to study the light-capturing properties of retinal in recently designed human cellular retinol binding protein II (hCRBPII) variants (Wang et al. Science, 2012, 338, 1340-1343). Our calculations show that these proteins absorb across a large part of the visible spectrum by combined polarization and electrostatic effects. These effects stabilize the ground or excited state energy levels of the retinal by perturbing the Schiff-base or ß-ionone moieties of the chromophore, which in turn modulates the amount of charge transfer within the molecule. Based on the predicted tuning principles, we design putative in silico mutations that further shift the absorption properties of retinal in hCRBPII towards the ultraviolet and infrared regions of the spectrum.