Dependent Binding Research Articles

Microstructure dependent binding of pigment epithelium derived factor (PEDF) to type I collagen fibrils.

Pigment epithelium derived factor (PEDF) is a multifunctional extracellular protein. In addition to its known anti-angiogenic and neurotrophic roles in collagen rich tissues, PEDF is thought to be involved in collagen fibril assembly due to its sequence specific binding to the collagen fibril and high expression in regions of active bone formation. In order to image the presence of the protein on the fibrils, PEDF was recombinantly made with a strep tag (strep-PEDF) and then gold nanoparticles conjugated to streptavidin (AuNP) were used as a secondary tag. The gold nanoparticles were detected using phase imaging in tapping mode AFM to image where exogenous PEDF bound in rabbit femur. These findings demonstrate that PEDF binds heterogeneously in cortical rabbit femur. Exogenous PEDF binding was concentrated at areas between microstructures with highly aligned collagen fibrils. Binding was not observed on or within the collagen fibrils themselves.

B12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release

Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.

Solvent-dependent binding interactions of the organophosphate pesticide, chlorpyrifos (CPF), and its metabolite, 3,5,6-trichloro-2-pyridinol (TCPy), with Bovine Serum Albumin (BSA): A comparative fluorescence quenching analysis

Analysis of the interaction of pesticides and their metabolites with the cellular proteins has drawn considerable attention in past several years to understand the effect of pesticides on environment and mankind. In this study, we have investigated the binding interaction of Bovine Serum Albumin (BSA) with a widely used organophosphorous insecticide chlorpyrifos (CPF), and its stable metabolite, 3,5,6-trichloro-2-pyridinol (TCPy) to provide a comparative analysis of the two molecules by employing various spectroscopic techniques viz., UV–vis absorption, Circular Dichroism (CD), and Fluorescence spectroscopy. The fluorescence quenching studies of BSA emission in two different solvents viz., water and methanol in presence of CPF and TCPy have led to the revelation of several interesting facts about the pesticide-protein interaction. It has been found that both the molecules cause static quenching of BSA emission as seen from the Stern-Volmer constant (Ksv) irrespective of the solvent used for the analysis. While TCPy is a stronger quencher in water, it exhibits comparable quenching capacity with CPF in methanol. The solvent dependent differential binding interaction of the two molecules finally indicates possibility of diverse bio-distribution of the pesticides within human body. The UV–vis and CD spectra of BSA in presence of the test molecules have unravelled that the molecules formed ground state complex that are highly reversible in nature and have minimal effect on the protein secondary structure. Furthermore it is also understood that structural changes of BSA in presence of CPF is significantly higher compared to that in presence of TCPY.

IL-2 functionalized hydroxyethylstarch nanocapsules for targeting of human T cells with different IL-2 receptor affinities in vitro and in vivo

Abstract Cell type specific delivery of drugs by use of nanocapsules (NC) is a promising approach for induction of efficient anti-tumor immunotherapies. Here, we report the generation of IL-2 functionalized NC (HES-IL-2) to target CD25+ (IL-2 receptor α chain) T cells. In flow cytometry experiments HES-IL-2 exhibited an enhanced uptake by human CD25+ T cells compared to control capsules. The observed uptake was CD25 specific as CD25high T cells displayed a significantly enhanced NC incorporation compared to CD25− T cells with negligible internalization. In addition blocking of the IL-2 receptor dependent binding by an anti CD25 mAb significantly reduced the HES-IL-2 uptake by human CD25+ T cells. Furthermore, comparing and competitive studies of naïve CD25−, activated CD25+ and regulatory CD25high human T cells revealed a low incorporation of HES-IL-2 in naïve and a moderate and high uptake, respectively in activated or regulatory T cells. Additionally, in order to preferentially address different T cell populations by means of various IL-2 receptor affinities, we generated HES-IL-2 with distinct reduced amounts of IL-2 on their surface. Intriguingly, in contrast to naïve CD25− and activated CD25+ T cells, in which the NC uptake was clearly IL-2 dose-dependent we did not observe any differences when HES-IL-2 with a twofold and tenfold decreased amount of IL-2 were used for studies with regulatory CD25high T cell, indicating a high IL-2 sensitivity of regulatory T cells for a specific targeting. In addition, in vivo studies in human T cell reconstituted RAG2−/−γc−/− mice exhibited a significantly enhanced uptake of HES-D-IL-2 by CD25+ T cells. In summary, we generated IL-2 functionalized NC for targeting of human CD4+CD25+ T cells in vitro and in vivo.

AnnexinA2 inhibition suppresses ovarian cancer progression via regulating β-catenin/EMT.

AnnexinA2 is a member of the Annexin family that acts as a Ca2+-dependent phospholipid and membrane binding protein, which is associated with the survival and spread of multiple neoplasms. However, the function of AnnexinA2 in ovarian cancer progression remains unclear. In this study, we aimed to investigate the role and underlying molecular mechanism of AnnexinA2 in cell proliferation and invasion in ovarian cancer. We found that the mRNA expression of AnnexinA2 was upregulated in ovarian cancer tissues and cell lines. In the loss-of-function of AnnexinA2, β-catenin was indicated to be significantly suppressed and EMT constrained. Moreover, cell proliferation and invasion were both markedly inhibited by the downregulation of AnnexinA2. Additionally, the overexpression of β-catenin obviously reversed the effect of AnnexinA2 on EMT, and cell proliferation and invasion, indicating that AnnexinA2 suppression regulated EMT through controlling β-catenin. Taken together, this study showed that AnnexinA2 inhibition suppresses proliferation and invasion in ovarian cancer via β-catenin/EMT, proposing the potential role of AnnexinA2 in the prevention and treatment of ovarian cancer.

Calcium-Independent Activation of an Allosteric Network in Langerin by Heparin Oligosaccharides.

The C-type lectin receptor Langerin is a glycan-binding protein that serves as an uptake receptor on Langerhans cells and is essential for the formation of Birbeck granules. Whereas most Langerin ligands are recognized by a canonical Ca2+ -dependent binding site, heparins have been proposed to make additional contacts to a secondary, Ca2+ -independent site. Glycan array screening and biomolecular NMR spectroscopy were employed to investigate the molecular mechanism of these interactions. We observed that binding of heparin hexasaccharides to a secondary site did not require the presence of Ca2+ and activated a previously identified intradomain allosteric network of Langerin (thus far only associated with Ca2+ affinity and release). We propose a communication hub between these two binding sites, which sheds new light on modulatory functions of Langerin-heparin interactions.

With no interaction, knockdown of Apollon and MDR1 reverse the multidrug resistance of human chronic myelogenous leukemia K562/ADM cells.

Chemotherapy is the main treatment method for patients with chronic myeloid leukemia(CML) and has achieved marked results. However, the acquisition of multidrug resistance(MDR) has seriously affected the quality of life and survival rate of patients. The overexpression of the inhibitors of apoptosis proteins(IAPs) and the adenosine triphosphate(ATP)-dependent binding cassette(ABC) transporters are the two main causes of MDR. Apollon and MDR1 are the most important and representative members, respectively, among the IAPs and ABC transporters. In the present study, we investigated the role of Apollon and MDR1 in chemotherapy resistance and their mechanism of interaction. We respectively knocked down the expression of Apollon and MDR1 using short hairpin RNA(shRNA) in adriamycin(ADM) resistant human CML K562 cells and examined the drug sensitivity, the consequences with regard to ADM accumulation and the alterations in the expression of Apollon and MDR1. The expression levels of Apollon and MDR1 mRNA were higher in the K562/ADM cells compared with the parental K562 cells as determined by reverse transcription‑polymerase chain reaction (RT-PCR). The plasmids of Apollon and MDR1 shRNA were respectively stably transfected into K562/ADM cells using Lipofectamine2000. The transfection efficiency was detected by fluorescence microscopy. Cell Counting Kit-8 (CCK-8) assay revealed that Apollon or MDR1 knockdown significantly increased the chemosensitivity of the K562/ADM cells to ADM. Flow cytometric assay revealed that K562/ADM/shMDR1 cells exhibited a significantly increased intracellular accumulation of ADM, and that changes were not found in the K562/ADM/shApollon cells. Compared with the parental K562/ADM cells, a significantly decreased expression of Apollon mRNA and protein was determined in the K562/ADM/shApollon cells without affecting the expression of MDR1 as determined by RT-PCR and western blotting. Likewise, the expression levels of MDR1 mRNA and protein also markedly downregulated in the K562/ADM/shMDR1 cells had no effect on Apollon expression. Collectively, our findings demonstrated, for the first time, that downregulation of Apollon or MDR1 through stable transfection with the Apollon- or MDR1-targeting shRNA induced MDR reversal through respective inhibition of Apollon or MDR1 expression and function. However, the reversal mechanism of Apollon and MDR1 revealed no direct interaction with each other.

Macrophage inducible C-type lectin (Mincle) recognizes glycosylated surface (S)-layer of the periodontal pathogen Tannerella forsythia.

The oral pathogen Tannerella forsythia is implicated in the development of periodontitis, a common inflammatory disease that leads to the destruction of the gum and tooth supporting tissues, often leading to tooth loss. T. forsythia is a unique Gram-negative organism endowed with an elaborate protein O-glycosylation system that allows the bacterium to express a glycosylated surface (S)-layer comprising two high molecular weight glycoproteins modified with O-linked oligosaccharides. The T. forsythia S-layer has been implicated in the modulation of cytokine responses of antigen presenting cells, such as macrophages, that play a significant role during inflammation associated with periodontitis. The macrophage-inducible C-type lectin receptor (Mincle) is an FcRγ-coupled pathogen recognition receptor that recognizes a wide variety of sugar containing ligands from fungal and bacterial pathogens. In this study, we aimed to determine if Mincle might be involved in the recognition of T. forsythia S-layer and modulation of cytokine response of macrophages against the bacterium. Binding studies using recombinant Mincle-Fc fusion protein indicated a specific Ca2+-dependent binding of Mincle to T. forsythia S-layer. Subsequent experiments with Mincle-expressing and Mincle-knockdown macrophages revealed a role for Mincle/S-layer interaction in the induction of both pro- and anti-inflammatory cytokine secretion in macrophages stimulated with T. forsythia as well as its S-layer. Together, these studies revealed Mincle as an important macrophage receptor involved in the modulation of cytokine responses of macrophages against T. forsythia, and thus may play a critical role in orchestrating the host immune response against the bacterium.

Mechanisms of vitamin D in regulation of calcium binding protein

Calbindin 9K(CaBP-9k)and Calbindin 28K(CaBP-28k), vitamin D dependent calcium binding protein(CaBP), play an important role in the the process of active transport of calcium.Research has shown that vitamin D can increase the expression of calcium binding protein, which can promote the absorption of calcium ions.Vitamin D deficiency and calcium binding protein could cause disorders of calcium homeostasis, such as hypocalcemia, rickets, osteoporosis and many other diseases.This review summarizes the studies of structure and distribution of CaBP and the mechanism of vitamin D in regulation of CaBP, which might provide new strategies for the prevention and treatment of paraplasia ossium. Key words: Calbinding; Vitamin D; Calcium; Transport

BabA dependent binding of Helicobacter pylori to human gastric mucins cause aggregation that inhibits proliferation and is regulated via ArsS

Mucins in the gastric mucus layer carry a range of glycan structures, which vary between individuals, can have antimicrobial effect or act as ligands for Helicobacter pylori. Mucins from various individuals and disease states modulate H. pylori proliferation and adhesin gene expression differently. Here we investigate the relationship between adhesin mediated binding, aggregation, proliferation and adhesin gene expression using human gastric mucins and synthetic adhesin ligand conjugates. By combining measurements of optical density, bacterial metabolic activity and live/dead stains, we could distinguish bacterial aggregation from viability changes, enabling elucidation of mechanisms behind the anti-prolific effects that mucins can have. Binding of H. pylori to Leb-glycoconjugates inhibited the proliferation of the bacteria in a BabA dependent manner, similarly to the effect of mucins carrying Leb. Furthermore, deletion of arsS lead to a decrease in binding to Leb-glycoconjugates and Leb-decorated mucins, accompanied by decreased aggregation and absence of anti-prolific effect of mucins and Leb-glycoconjugates. Inhibition of proliferation caused by adhesin dependent binding to mucins, and the subsequent aggregation suggests a new role of mucins in the host defense against H. pylori. This aggregating trait of mucins may be useful to incorporate into the design of adhesin inhibitors and other disease intervention molecules.

Optical peak gain in a PbSe/CdSe core-shell quantum dot in the presence of magnetic field for mid-infrared laser applications

Magnetic field induced electronic properties and some nonlinear optical properties of an exciton are investigated taking into account the geometrical confinement effects in a PbSe/CdSe core-shell heterostructure. The effects of dielectric mismatch between the inner and outer materials, built-in internal electric fields and the self-polarization potential are included in the calculation of obtaining these properties. Self-polarization charges are incorporated at the interface of the core-shell materials. The magnetic field dependent exciton binding energies are found with the ratio of core-shell radii of the dot using variational formulism within the single band effective mass approximation. The magnetic field induced oscillator strength and the radiative life time of the exciton are studied for a constant dot radius. The absorption coefficients and the optical gain as a function of photon energy, the peak gain with the current density are investigated in the PbSe/CdSe core-shell dot for various values of magnetic field strength.

Differential Pattern of Arsenic Binding by the Cell Wall in Two ArseniteTolerant Bacillus Strains Isolated from Arsenic Contaminated Soil

Arsenite binding was evaluated in two Bacillus strains i.e., B. megaterium and B. pumilus, isolated from arsenic contaminated soil of Unnao district of Uttar Pradesh (India). Initial results showed that more than 90% of arsenite was removed by surface binding by the cell wall component in both the tested species of bacteria. Results on the concentration dependent arsenic binding in bacterial strains exhibited higher efficiency of arsenite binding in B. megaterium (qmax - 1000 mg g-1 protein) than B. pumilus (qmax - 666.7 mg g-1 protein). The pH optima for arsenic (As) binding in both B. megaterium (pH 6.0) and B. pumilis (pH 8.0) were found to be different. Results on temperature dependent arsenite binding by B. megaterium showed maximum binding at 30oC, while arsenic binding maxima in B. pumilus showed a broad temperature range (25oC to 35oC). The kinetic parameters on arsenite binding revealed that both the bacterial strains followed pseudo-second order kinetics. The As adsorption behavior of the bacterial strains was better explained by Langmuir isotherm rather than Freundlich model. Results of FTIR spectra on surface binding of As revealed major spectral changes in the band region of 1600 cm-1 to 800 cm-1 in case of B. megaterium, indicating involvement of mainly amines, alkenes and C-N functional groups. Whereas FTIR spectrum of B. pumilus showed changes in the band region of 3433 cm-1 to 2924 cm-1 , indicating the involvement of hydroxyl, alkanes, alkenes, amides and aromatic functional groups in the arsenic binding. A corollary of these results indicated differential binding of arsenite in both the Bacillus strains was on account of different arsenite binding ligands on cell surface as evident from the FTIR results as well as different pH and temperature optima.

The upregulation of annexin A2 after spinal cord injury in rats may have implication for astrocyte proliferation

Annexin A2 (ANXA2), is a member of the annexin family of proteins that exhibit Ca2+-dependent binding to phospholipids. One attractive biological function of ANXA2 is participating in DNA synthesis and cell proliferation. Previous studies have shown that ANXA2 play a role in the development of the central nervous system. However, the biological function of ANXA2 after spinal cord injury (SCI) is still with limited acquaintance. In the present study, we performed a SCI model in adult rats and investigated the dynamic changes of ANXA2 expression in the spinal cord. Western blot analysis indicated a striking expression upregulation of ANXA2 after SCI. Immunohistochemistry further confirmed that ANXA2 immunoactivity was expressed at low levels in normal condition and increased at 5day after SCI. Double immunofluorescence staining prompted that ANXA2 immunoreactivity was found in astrocytes and neurons. Interestingly, ANXA2 expression was increased predominantly in astrocytes. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA), Cyclin D1 and active caspase-3 in the injured spinal cords by western blot. Co-expression of ANXA2/PCNA, ANXA2/Cyclin D1 was detected in glial fibrillary acidic protein. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many ANXA2-expressing cells and rare caspase-3 was observed in ANXA2-expressing cells after SCI. In addition, ANXA2 knockdown in astrocytes resulted in the increase of PCNA expression after LPS stimulation, showing that ANXA2 inhibited astrocyte proliferation after inflammation. Our data suggested that ANXA2 might play important roles in CNS pathophysiology after SCI.

Purification of Monoclonal Antibodies Using a Fiber Based Cation-Exchange Stationary Phase: Parameter Determination and Modeling.

Monoclonal antibodies (mAb) currently dominate the market for protein therapeutics. Because chromatography unit operations are critical for the purification of therapeutic proteins, the process integration of novel chromatographic stationary phases, driven by the demand for more economic process schemes, is a field of ongoing research. Within this study it was demonstrated that the description and prediction of mAb purification on a novel fiber based cation-exchange stationary phase can be achieved using a physico-chemical model. All relevant mass-transport phenomena during a bind and elute chromatographic cycle, namely convection, axial dispersion, boundary layer mass-transfer, and the salt dependent binding behavior in the fiber bed were described. This work highlights the combination of model adaption, simulation, and experimental parameter determination through separate measurements, correlations, or geometric considerations, independent from the chromatographic cycle. The salt dependent binding behavior of a purified mAb was determined by the measurement of adsorption isotherms using batch adsorption experiments. Utilizing a combination of size exclusion and protein A chromatography as analytic techniques, this approach can be extended to a cell culture broth, describing the salt dependent binding behavior of multiple components. Model testing and validation was performed with experimental bind and elute cycles using purified mAb as well as a clarified cell culture broth. A comparison between model calculations and experimental data showed a good agreement. The influence of the model parameters is discussed in detail.

Cadmium-free aqueous synthesis of ZnSe and ZnSe@ZnS core–shell quantum dots and their differential bioanalyte sensing potential

Herein we report a facile and cadmium-free approach to prepare water-soluble fluorescent ZnSe@ZnS core–shell quantum dots (QDs), using thioglycolic acid (TGA) ligand as a stabilizer and thiourea as a sulfur source. The optical properties and morphology of the obtained core–shell QDs were characterized by UV–vis and fluorescence spectroscopy, transmission electron microscopy (TEM), energy-dispersive x-ray analysis (EDX), x-ray diffraction (XRD), electrophoresis and dynamic light scattering (DLS) techniques. TEM analysis, and electrophoresis data showed that ZnSe core had an average size of 3.60 ± 0.12 nm and zeta potential of −38 mV; and for ZnSe@ZnS QDs, the mean size was 4.80 ± 0.20 nm and zeta potential was −45 mV. Compared to the core ZnSe QDs, the quantum yield of these core–shell structures was higher (13% versus 32%). These were interacted with five common bioanalytes such as, ascorbic acid, citric acid, oxalic acid, glucose and cholesterol which revealed fluorescence quenching due to concentration dependent binding of analytes to the core only, and core–shell QDs. The binding pattern followed the sequence: cholesterol < glucose < ascorbic acid < oxalic acid < citric acid for ZnSe, and cholesterol < glucose < oxalic acid < ascorbic acid < citric acid for core–shell QDs. Thus, enhanced binding was noticed for the analyte citric acid which may facilitate development of a fluorescence-based sensor based on the ZnSe core-only quantum dot platform. Further, the hydrophilic core–shell structure may find use in cell imaging applications.

Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter.

Neurotransmitter/sodium symporters (NSSs) are responsible for Na+-dependent reuptake of neurotransmitters and represent key targets for antidepressants and psychostimulants. LeuT, a prokaryotic NSS protein, constitutes a primary structural model for these transporters. Here we show that K+ inhibits Na+-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K+-induced conformational dynamics we measured fluorescence resonance energy transfer (FRET) between fluorescein site-specifically attached to inserted cysteines and Ni2+ bound to engineered di-histidine motifs (transition metal ion FRET). The measurements supported K+-induced closure of the transporter to the outside, which was counteracted by Na+ and substrate. Promoting an outward-open conformation of LeuT by mutation abolished the K+-effect. The K+-effect depended on an intact Na1 site and mutating the Na2 site potentiated K+ binding by facilitating transition to the inward-facing state. The data reveal an unrecognized ability of K+ to regulate the LeuT transport cycle.

Surface plasmon resonance and isothermal titration calorimetry to monitor the Ni(II)-dependent binding of Helicobacter pylori NikR to DNA.

NikR is a transcription factor that regulates the expression of Ni(II)-dependent enzymes and other proteins involved in nickel trafficking. In the human pathogenic bacterium Helicobacter pylori, NikR (HpNikR) controls, among others, the expression of the Ni(II) enzyme urease by binding the double-strand DNA (dsDNA) operator region of the urease promoter (OP ureA ) in a Ni(II)-dependent mode. This article describes the complementary use of surface plasmon resonance (SPR) spectroscopy and isothermal titration calorimetry (ITC) to carry out a mechanistic characterization of the HpNikR-OP ureA interaction. An active surface was prepared by affinity capture of OP ureA and validated for the recognition process in the SPR experiments. Subsequently, the Ni(II)-dependent affinity of the transcription factor for its operator region was assessed through kinetic evaluation of the binding process at variable Ni(II) concentrations. The kinetic data are consistent with a two-step binding mode involving an initial encounter between the two interactants, followed by a conformational rearrangement of the HpNikR-OP ureA complex, leading to high affinity binding. This conformational change is only observed in the presence of the full set of four Ni(II) ions bound to the protein. The SPR assay developed and validated in this study constitutes a suitable method to screen potential drug lead candidates acting as inhibitors of this protein-dsDNA interaction. Graphical Abstract Pictorial representation of the interaction between HpNikR, flowing in solution, and the OP ureA urease promoter immobilized on the sensor chip surface.

Abstract 1928: Comprehensive identification of miR-122 targetome in the liver and HCC by HITS-CLIP analysis reveals conserved regulation of AMPK signaling in hepatocarcinogenesis

Abstract miR-122, a conserved liver-specific miRNA, is critical for maintaining liver homeostasis. Loss of miR-122 is associated with loss of hepatic phenotype, metastasis and poor prognosis. Recent in vivo work has established miR-122 as a bona fide tumor suppressor, with poorly differentiated, Afp-positive, high grade, spontaneous HCC observed in liver-specific (LKO) and germ-line (KO) miR-122 knockout mice. We hypothesized that the pathophysiology exhibited by KO mice would be mediated through de-repressed targets in KO livers and therefore, dysfunctional regulation of specific pathways involved in liver homeostasis and carcinogenesis. Although the role of miR-122 in the maintenance of liver homeostasis is well established, systematic and direct biochemical elucidation of the miR-122 target network remains incomplete. To this end, we performed Ago-dHITS-CLIP analysis in livers of 6 week-old wild type (WT) and KO mice to identify miRNA targetome in vivo, which revealed ∼1800 miR-122 binding sites, with over 30 fold enrichment for the miR-122 seed sequence sites in WT compared to KO livers. Surprisingly, a majority of miR-122 binding sites were observed on coding exons (40%) followed by 3’-UTRs, introns and 5’-UTRs. Motif analysis of miR-122 dependent binding sites lacking the seed sequence revealed a novel G-bulged motif in addition to the canonical miR-122 seed sequences. Moreover, the hepatic gene expression profile of these mice measured by RNA-seq showed up regulation of ∼2500 genes and down regulation of ∼1200 genes in KO livers compared to WT livers. A comparison of HITS-CLIP and RNA-seq data revealed that majority of miR-122 targets harboring canonical binding sites at their 3’UTRs as well as some coding regions, are significantly upregulated in miR-122 KO livers without notable alteration in the expression of or G-bulged targets at the RNA level. HITS-CLIP analysis of several human HCC and matching benign liver tissues also identified both canonical and G-bulged targets; where the majority of both are located in the coding regions followed by 3’UTRs. Association of miR-122 targets with Argonaute was precipitously reduced in HCCs expressing lower miR-122 levels than respective benign livers. Notably, though miR-122 is completely conserved and highly expressed in human and mouse livers, a large number of identified targets seem to be species-specific. Ingenuity Pathway Analysis of enriched targets revealed conserved pathway specific regulation in mouse and human livers. Pathways commonly associated with critical roles in liver carcinogenesis and liver metabolism, such as AMPK signaling and PI3/AKT pathways, were the major targets of miR-122-mediated regulation. This study suggests that miR-122 functionality is conserved across species. Citation Format: Juan Barajas. Comprehensive identification of miR-122 targetome in the liver and HCC by HITS-CLIP analysis reveals conserved regulation of AMPK signaling in hepatocarcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1928.

Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein.

The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg2+ coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg2+-dependent sterol binding by Pry1.

Phosphorylation of the C-terminal tail of proteasome subunit α7 is required for binding of the proteasome quality control factor Ecm29.

The proteasome degrades many short-lived proteins that are labeled with an ubiquitin chain. The identification of phosphorylation sites on the proteasome subunits suggests that degradation of these substrates can also be regulated at the proteasome. In yeast and humans, the unstructured C-terminal region of α7 contains an acidic patch with serine residues that are phosphorylated. Although these were identified more than a decade ago, the molecular implications of α7 phosphorylation have remained unknown. Here, we showed that yeast Ecm29, a protein involved in proteasome quality control, requires the phosphorylated tail of α7 for its association with proteasomes. This is the first example of proteasome phosphorylation dependent binding of a proteasome regulatory factor. Ecm29 is known to inhibit proteasomes and is often found enriched on mutant proteasomes. We showed that the ability of Ecm29 to bind to mutant proteasomes requires the α7 tail binding site, besides a previously characterized Rpt5 binding site. The need for these two binding sites, which are on different proteasome subcomplexes, explains the specificity of Ecm29 for proteasome holoenzymes. We propose that alterations in the relative position of these two sites in different conformations of the proteasome provides Ecm29 the ability to preferentially bind specific proteasome conformations.