Upstream stimulatory factor‐2 mediates quercetin‐induced suppression of PAI‐1 gene expression in human endothelial cells

The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor‐1 (PAI‐1) in cultured endothelial cells (ECs). Transfection of PAI‐1 promoter‐luciferase reporter deletion constructs identified a 251‐bp fragment (nucleotides ?800 to ?549) responsive to Quer. Two E‐box motifs (CACGTG), at map positions ?691 (E‐box1) and ?575 (E‐box2), are platforms for occupancy by several members of the c‐MYC family of basic helix‐loop‐helix leucine zipper (bHLH‐LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)‐1 and USF‐2 as E‐box1/E‐box2 binding factors. ECs co‐transfected with a 251 bp PAI‐1 promoter fragment containing the two E‐box motifs (p251/luc) and a USF‐2 expression vector (pUSF‐2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF‐2 decreased, while transfection of a dominant‐negative USF construct increased, EC growth consistent with the known anti‐proliferative properties of USF proteins. Quer‐induced decreases in PAI‐1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols. J. Cell. Biochem. 111: 720–726, 2010. © 2010 Wiley‐Liss, Inc.     

Study on structure and assembly of the third transmembrane domain of Slc11a1

The structure and self‐assembly of the peptide corresponding to the third transmembrane domain (TMD3) of Slc11a1 and its E139A mutant are studied in 1,1,1,3,3,3‐hexafluoro‐2‐propanol (HFIP) aqueous solution by NMR and CD experiments. Slc11a1 is an integral membrane protein with 12 putative TMDs and functions as a pH‐coupled divalent metal cation transporter. Glu139 of Slc11a1 is highly conserved within predicted TMD3 of the Slc11 protein family and function‐associated. Here, we provide the first direct experimental evidence for the structural features of two 24‐residue peptides corresponding to TMD3 of Slc11a1 and its E139A mutant in 60% HFIP‐d₂ aqueous solution using CD and NMR spectroscopies. Our study shows that the membrane‐spanning peptide folds as a typical amphipathic α‐helix structure from Ile5 to Met20 with hydrophilic residues Glu12 (Glu139 in Slc11a1) and Asp19 lying on the same side of the helix. The substitution of Glu139 by an alanine residue has little effect on the structure of the peptide, but increases hydrophobicity and facilitates self‐assembly of the peptide. Although the wildtype peptide is monomeric in HFIP aqueous solution, the E139A mutant forms a dimer. The increase in hydrophobicity of the membrane‐spanning peptide and/or change in the interactions between transmembrane segments induced by E139A mutation may affect the metal ion transport of the protein. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

RANBP2 is an allosteric activator of the conventional kinesin‐1 motor protein,KIF5B,in a minimal cell‐free system

The association of cargoes to kinesins is thought to promote kinesin activation, yet the validation of such a model with native cargoes is lacking because none is known to activate kinesins directly in an in vitro system of purified components. The RAN‐binding protein 2 (RANBP2), through its kinesin‐binding domain (KBD), associates in vivo with kinesin‐1, KIF5B/KIF5C. Here, we show that KBD and its flanking domains, RAN GTPase‐binding domains 2 and 3 (RBD2/RBD3), activate the ATPase activity of KIF5B approximately 30‐fold in the presence of microtubules and ATP. The activation kinetics of KIF5B by RANBP2 is biphasic and highly cooperative. Deletion of one of its RBDs lowers the activation of KIF5B threefold and abolishes cooperativity. Remarkably, RBD2–KBD–RBD3 induces unfolding and modest activation of KIF5B in the absence of microtubules. Hence, RANBP2 is the first native and positive allosteric activator known to jump‐start and boost directly the activity of a kinesin.     

RANBP2 and USP9x regulate nuclear import of adenovirus minor coat protein IIIa

As intracellular parasites, viruses exploit cellular proteins at every stage of infection. Adenovirus outbreaks are associated with severe acute respiratory illnesses and conjunctivitis, with no specific antiviral therapy available. An adenoviral vaccine based on human adenovirus species D (HAdV-D) is currently in use for COVID-19. Herein, we investigate host interactions of HAdV-D type 37 (HAdV-D37) protein IIIa (pIIIa), identified by affinity purification and mass spectrometry (AP-MS) screens. We demonstrate that viral pIIIa interacts with ubiquitin-specific protease 9x (USP9x) and Ran-binding protein 2 (RANBP2). USP9x binding did not invoke its signature deubiquitination function but rather deregulated pIIIa-RANBP2 interactions. In USP9x-knockout cells, viral genome replication and viral protein expression increased compared to wild type cells, supporting a host-favored mechanism for USP9x. Conversely, RANBP2-knock down reduced pIIIa transport to the nucleus, viral genome replication, and viral protein expression. Also, RANBP2-siRNA pretreated cells appeared to contain fewer mature viral particles. Transmission electron microscopy of USP9x-siRNA pretreated, virus-infected cells revealed larger than typical paracrystalline viral arrays. RANBP2-siRNA pretreatment led to the accumulation of defective assembly products at an early maturation stage. CRM1 nuclear export blockade by leptomycin B led to the retention of pIIIa within cell nuclei and hindered pIIIa-RANBP2 interactions. In-vitro binding analyses indicated that USP9x and RANBP2 bind to C-terminus of pIIIa amino acids 386–563 and 386–510, respectively. Surface plasmon resonance testing showed direct pIIIa interaction with recombinant USP9x and RANBP2 proteins, without competition. Using an alternative and genetically disparate adenovirus type (HAdV-C5), we show that the demonstrated pIIIa interaction is also important for a severe respiratory pathogen. Together, our results suggest that pIIIa hijacks RANBP2 for nuclear import and subsequent virion assembly. USP9x counteracts this interaction and negatively regulates virion synthesis. This analysis extends the scope of known adenovirus-host interactions and has potential implications in designing new antiviral therapeutics.     

Spectroscopic and molecular modelling analysis of the interaction between ethane‐1,2‐diyl bis(N,N‐dimethyl‐N‐hexadecylammoniumacetoxy)dichloride and bovine serum albumin

Several spectroscopic approaches namely fluorescence, time‐resolved fluorescence, UV‐visible, and Fourier transform infra‐red (FT‐IR) spectroscopy were employed to examine the interaction between ethane‐1,2‐diyl bis(N,N‐dimethyl‐N‐hexadecylammoniumacetoxy)dichloride (16‐E2‐16) and bovine serum albumin (BSA). Fluorescence studies revealed that 16‐E2‐16 quenched the BSA fluorescence through a static quenching mechanism, which was further confirmed by UV–visible and time‐resolved fluorescence spectroscopy. In addition, the binding constant and the number of binding sites were also calculated. The thermodynamic parameters at different temperatures (298 K, 303 K, 308 K and 313 K) indicated that 16‐E2‐16 binding to BSA is entropy driven and that the major driving forces are electrostatic interactions. Decrease of the α‐helix from 53.90 to 46.20% with an increase in random structure from 22.56 to 30.61% were also observed by FT‐IR. Furthermore, the molecular docking results revealed that 16‐E2‐16 binds predominantly by electrostatic and hydrophobic forces to some residues in the BSA sub‐domains IIA and IIIA. Copyright © 2015 John Wiley & Sons, Ltd.     

Two new miR‐16 targets: caprin‐1 and HMGA1, proteins implicated in cell proliferation

Background information. miRNAs (microRNAs) are a class of non‐coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3′ UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR‐16 (miRNA‐16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR‐16. Results. In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR‐16, caprin‐1 (cytoplasmic activation/proliferation‐associated protein‐1) and HMGA1 (high‐mobility group A1), and we also studied cyclin E which had been previously recognized as an miR‐16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR‐16 interacts with the 3′ UTR of the three target mRNAs. We showed that miR‐16, in MCF‐7 and HeLa cell lines, down‐regulates the expression of caprin‐1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. Conclusions. Taken together, our data demonstrated that miR‐16 can negatively regulate two new targets, HMGA1 and caprin‐1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.     

Structural scaffold for eIF4E binding selectivity of 4E‐BP isoforms: crystal structure of eIF4E binding region of 4E‐BP2 and its comparison with that of 4E‐BP1

To clarify the higher eukaryotic initiation factor 4E (eIF4E) binding selectivity of 4E‐binding protein 2 (4E‐BP2) than of 4E‐BP1, as determined by Trp fluorescence analysis, the crystal structure of the eIF4E binding region of 4E‐BP2 in complex with m⁷GTP‐bound human eIF4E has been determined by X‐ray diffraction analysis and compared with that of 4E‐BP1. The crystal structure revealed that the Pro47‐Ser65 moiety of 4E‐BP2 adopts a L ‐shaped conformation involving extended and α‐helical structures and extends over the N‐terminal loop and two different helix regions of eIF4E through hydrogen bonds, and electrostatic and hydrophobic interactions; these features were similarly observed for 4E‐BP1. Although the pattern of the overall interaction of 4E‐BP2 with eIF4E was similar to that of 4E‐BP1, a notable difference was observed for the 60–63 sequence in relation to the conformation and binding selectivity of the 4E‐BP isoform, i.e. Met‐Glu‐Cys‐Arg for 4E‐BP1 and Leu‐Asp‐Arg‐Arg for 4E‐BP2. In this paper, we report that the structural scaffold of the eIF4E binding preference for 4E‐BP2 over 4E‐BP1 is based on the stacking of the Arg63 planar side chain on the Trp73 indole ring of eIF4E and the construction of a compact hydrophobic space around the Trp73 indole ring by the Leu59‐Leu60 sequence of 4E‐BP2. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.