Two minute video looks at the differences between Repeat Signage Standard edition and the Standalone edition. The former has full features and is ideal for displaying engaging content on any display screen anywhere in the world, whilst the latter is ideal for any application where the Internet is not possible, for example, military, army manoevers, evacuation, outdoor events, etc.

Repeat Signage software celebrates 12 years with the launch of Version 5 and a new licencing model. Repeat Signage V5 (Version 5), which supports Windows 10 and 11, has new and improved features including the ability to link with a user’s eBay account to display eBay auctions on screen.

7-minute video shows you how to filter and display data in a datagrid as part of your digital signage software presentations for display on any screen, anywhere. This feature is available in Repeat Signage Corporate or Media Wall editions and is ideal for corporate offices to help keep staff updated on relevant information

The spreadsheet control within Repeat Signage software allows you to display existing Microsoft Excel .xlsx files and allows you to use the built in editor to create spreadsheets within your presentations. You can also create and display spreadsheet graphs from your information. This gives you an easy way to display formatted information in grids and you also have the ability to insert pictures as well.

7-minute video shows you how to use the Repeat Signage designer screen to insert controls, e.g. picture control, to create flexible, dynamic, digital signage presentations for display screens.

In order for your business to have a professional presence online, you must seek the services of a...

The post 5 skills your web designer should have appeared first on Ignition Media.

As a small business owner, regardless of what business you are in, you need to have a great...

The post Why It Pays to Invest in your Website Design appeared first on Ignition Media.

At Ignition Media, as we embrace 2024, we’re keenly aware of the evolving trends in web design that...

The post The Top Website Design Trends for 2024 appeared first on Ignition Media.

The following article, 7 Bad Signs for Democrats: A Satirical Take on America’s Return to Normalcy, was first published on The Black Sphere.

Yes, the Democrats got shellacked in the last election. And while that’s a breath of fresh air, it’s hardly the only sign that the political winds are changing.

Continue reading 7 Bad Signs for Democrats: A Satirical Take on America’s Return to Normalcy ...

By David Blackmon In the wake of the election of President Donald Trump to serve a second term in office, along with presumptive Republican majorities in both houses of Congress, many are now asking about what the future will hold for the oddly named Inflation Reduction Act. Trump made it repeatedly clear on the campaign […]

The post Biden’s Signature Climate ‘Boondoggle’ Might Be On Chopping Block After Trump Win appeared first on Liberty Unyielding.

Gabriel Bortoleto, 20, will make his Formula One debut for the Audi-owned Sauber next season.

2 ‘Moody’ Home Design Trends Generating Buzz

Justin Welby has stepped down over a church abuse scandal.

The Archbishop of Canterbury has announced he will step down over a damning report into abuse.

People are angry at the authorities' handling of floods which killed more than 200 people.

Joe Biden’s corrupt Federal Emergency Management Administration (FEMA) has been caught telling its on-the-ground operatives not to help anyone with a Donald Trump campaign sign in their yard. The news broke late last week when a whistleblower revealed agency messages that told workers to refuse to help Trump supporters in the wake of Hurricane Milton […]

The post Biden’s Corrupt FEMA Told Workers Not to Help Hurricane Victims Who Had Trump Signs appeared first on The Lid.

The leader of the world's Anglican communion, Archbishop of Canterbury Justin Welby, announced Tuesday he was resigning following a damning report that concluded the Church of England covered up a serial abuse case.

The ARJ21 is now to be known as the C909. The renaming plays into COMAC's ambition to enter the global market.

The following article, Biden’s Corrupt FEMA Told Workers Not to Help Hurricane Victims Who Had Trump Signs, was first published on Conservative Firing Line.

Joe Biden’s corrupt Federal Emergency Management Administration (FEMA) has been caught telling its on-the-ground operatives not to help anyone with a Donald Trump campaign sign in their yard. The news broke late last week when a whistleblower revealed agency messages that told workers to refuse to help Trump supporters in the wake of Hurricane Milton …

Continue reading Biden’s Corrupt FEMA Told Workers Not to Help Hurricane Victims Who Had Trump Signs ...

Russia–China defence and security relations: Insights from the expert community 13 September 2022 — 3:00PM TO 4:00PM Anonymous (not verified) 31 August 2022 Online

Experts share insights on Russia–China military, defence, and security relations. 

In the defence and security realms, Russia–China relations resemble more pragmatic cooperation based on shared, calculated interests than an alliance.

This event presents and discusses key findings from a recent expert survey conducted by Chatham House with the aim to gather insights on Russia–China military, defence, and security relations.

Survey responses helped identify areas of bilateral cooperation but also crucial friction points and obstacles that prevent the relationship from developing further, as well as policy pathways for the West.

Heterotrimeric G-proteins are signaling switches broadly divided into four families based on the sequence and functional similarity of their Gα subunits: Gs, Gi/o, Gq/11, and G12/13. Artificial mutations that activate Gα subunits of each of these families have long been known to induce oncogenic transformation in experimental systems. With the advent of next-generation sequencing, activating hotspot mutations in Gs, Gi/o, or Gq/11 proteins have also been identified in patient tumor samples. In contrast, patient tumor-associated G12/13 mutations characterized to date lead to inactivation rather than activation. By using bioinformatic pathway analysis and signaling assays, here we identified cancer-associated hotspot mutations in Arg-200 of Gα13 (encoded by GNA13) as potent activators of oncogenic signaling. First, we found that components of a G12/13-dependent signaling cascade that culminates in activation of the Hippo pathway effectors YAP and TAZ is frequently altered in bladder cancer. Up-regulation of this signaling cascade correlates with increased YAP/TAZ activation transcriptional signatures in this cancer type. Among the G12/13 pathway alterations were mutations in Arg-200 of Gα13, which we validated to promote YAP/TAZ-dependent (TEAD) and MRTF-A/B-dependent (SRE.L) transcriptional activity. We further showed that this mechanism relies on the same RhoGEF-RhoGTPase cascade components that are up-regulated in bladder cancers. Moreover, Gα13 Arg-200 mutants induced oncogenic transformation in vitro as determined by focus formation assays. In summary, our findings on Gα13 mutants establish that naturally occurring hotspot mutations in Gα subunits of any of the four families of heterotrimeric G-proteins are putative cancer drivers.

Hdac3 is a lysine deacetylase that removes acetyl groups from histones and additional proteins. Although Hdac3 functions within mesenchymal lineage skeletal cells are defined, little is known about Hdac3 activities in bone-resorbing osteoclasts. In this study we conditionally deleted Hdac3 within Ctsk-expressing cells and examined the effects on bone modeling and osteoclast differentiation in mice. Hdac3 deficiency reduced femur and tibia periosteal circumference and increased cortical periosteal osteoclast number. Trabecular bone was likewise reduced and was accompanied by increased osteoclast number per trabecular bone surface. We previously showed that Hdac3 deacetylates the p65 subunit of the NF-κB transcriptional complex to decrease DNA-binding and transcriptional activity. Hdac3-deficient osteoclasts demonstrate increased K310 NF-κB acetylation and NF-κB transcriptional activity. Hdac3-deficient osteoclast lineage cells were hyper-responsive to RANKL and showed elevated ex vivo osteoclast number and size and enhanced bone resorption in pit formation assays. Osteoclast-directed Hdac3 deficiency decreased cortical and trabecular bone mass parameters, suggesting that Hdac3 regulates coupling of bone resorption and bone formation. We surveyed a panel of osteoclast-derived coupling factors and found that Hdac3 suppression diminished sphingosine-1-phosphate production. Osteoclast-derived sphingosine-1-phosphate acts in paracrine to promote bone mineralization. Mineralization of WT bone marrow stromal cells cultured with conditioned medium from Hdac3-deficient osteoclasts was markedly reduced. Expression of alkaline phosphatase, type 1a1 collagen, and osteocalcin was also suppressed, but no change in Runx2 expression was observed. Our results demonstrate that Hdac3 controls bone modeling by suppressing osteoclast lineage cell responsiveness to RANKL and coupling to bone formation.

Ligand bias is the ability of ligands to differentially activate certain receptor signaling responses compared with others. It reflects differences in the responses of a receptor to specific ligands and has implications for the development of highly specific therapeutics. Whereas ligand bias has been studied primarily for G protein–coupled receptors (GPCRs), there are also reports of ligand bias for receptor tyrosine kinases (RTKs). However, the understanding of RTK ligand bias is lagging behind the knowledge of GPCR ligand bias. In this review, we highlight how protocols that were developed to study GPCR signaling can be used to identify and quantify RTK ligand bias. We also introduce an operational model that can provide insights into the biophysical basis of RTK activation and ligand bias. Finally, we discuss possible mechanisms underpinning RTK ligand bias. Thus, this review serves as a primer for researchers interested in investigating ligand bias in RTK signaling.

The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)–linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants R521G and P525L associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA-binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.

Yi Zhang
Sep 1, 2005; 4:1240-1250
Research

Joris J. Benschop
Jul 1, 2007; 6:1198-1214
Research

Albrecht Gruhler
Mar 1, 2005; 4:310-327
Research

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four Cα-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-α-carbon thioether–containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by Cα H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an α,β-dehydro-amino acid intermediate during Cα-thioether bridge LC–MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.

The break-up of Scholz’s coalition government signals the end of Germany’s old economic model Expert comment jon.wallace 12 November 2024

The coalition could not agree how to fund new support for Ukraine and failed to fully implement the ‘Zeitenwende’. A new government must push through reform.

As Europeans were still processing Donald Trump’s victory in the 2024 US presidential election, an acrimonious break up occurred 4000 miles east of Washington DC.

Reports had been circulating for weeks about the fragile state of Germany’s ‘traffic light’ coalition government led by German Chancellor Olaf Scholz, consisting of the centre-left Social Democratic Party (SPD), the Green Party, and liberal Free Democratic Party (FDP).

The expectation had been that the coalition would hold on for a few more weeks and might even be given a new lease of life by Trump’s re-election. Instead, it collapsed on the day Trump’s win was confirmed. An unusually angry Scholtz announced in a live address that he had fired FDP Finance Minister Christian Lindner, effectively breaking up the coalition.

At the heart of the dispute was the so-called ‘debt brake’ – a constitutional mechanism which restricts Germany’s annual public deficit to 0.35 per cent of GDP. Lindner proposed a set of reforms which were unpalatable to the SPD and the Greens. 

In response, Scholz suggested declaring an emergency, which would have suspended the debt brake. That in turn was unacceptable to Lindner, leading to his sacking by the Chancellor.

Practically, this means the SPD and the Greens are now in a minority coalition, without agreement on the 2025 budget or the votes in parliament to pass it. They also still face the challenge of the debt brake.

A vote of confidence will take place in December, with elections to be held before the end of February 2025 latest.

The end of Germany’s economic model

At the root of Germany’s political crisis is the country’s economic model. For decades, Germany relied on a system that depended on cheap Russian gas, cheap imports of consumer goods from China, high-value exports – particularly in the automotive sector – and the US security umbrella.

With Russian energy no longer viable, the global economic landscape shifting, and Donald Trump on his way back to the White House, that model is no longer workable. And Germany’s economy is expected to contract by 0.2 per cent in 2024 – a contraction for the second year running.

The ‘Zeitenwende’, announced by Scholz in the wake of Russia’s full-scale invasion of Ukraine, should have signalled a turnaround of both foreign and economic policy, given how much the two are interconnected. Yet on both fronts, too little changed.

Germany’s reliance on Russian gas did come to an abrupt end in 2022. And Germany is Ukraine’s second largest military aid donor after the US, while accepting the most Ukrainian refugees.

But the ‘Zeitenwende’ turnaround ended there. Scholz’s coalition government failed to prepare for long-term investment in defence at the levels required by creating an off-budget defence spending fund which would have run out in 2027. The draft budget for 2025 showed defence spending would have been cut, as would support for Ukraine.

Germany has also struggled to turn around its economic woes, with the car industry particularly affected. Cheap Chinese EVs and new energy technologies are competing with Germany’s most powerful companies. Volkswagen, the country’s largest car manufacturer, has announced plant closures and layoffs due to shrinking profit margins.  

To the west, Trump’s threat to impose 10 to 20 per cent tariffs on all EU imports meant share prices of Volkswagen, BMW, Mercedez-Benz and Porsche all dropped between 4 to 7 per cent following news of his re-election.

To the east, trade tensions between the EU and China are intensifying. Yet rather than choosing to diversify, German companies have doubled down on their bets in China, with German investment in the country rising from €6.5bn for the whole of 2023 to €7.3bn in the first half of 2024 alone – only exposing carmakers further.

Germany’s support for Ukraine

Like French President Emmanuel Macron, Scholz had already been weakened by the results of the European Parliamentary elections in June. With the collapse of his traffic light coalition, the EU’s Franco-German ‘engine’ is now well and truly stalled – until new leadership can be found. This weakness comes at a perilous moment when clear, united European leadership, and much increased funding, is needed to shore up support for Ukraine.

George H. Rothblat
May 1, 1999; 40:781-796
Reviews

Announcing Design Resonance in an Age of Crisis News Release sysadmin 1 June 2020

London Design Biennale and Chatham House announce Design Resonance in an Age of Crisis, which calls for action by designers around the world to create radical design solutions to critical problems across four key areas: Health, Environment, Society and Work.

Design In An Age of Crisis - Open Call News Release sysadmin 21 July 2020

Chatham House and London Design Biennale announce full details of 'Design In An Age of Crisis,' a global Open Call for radical design thinking.

Design in an Age of Crisis Launches News Release jon.wallace 13 January 2021

Design open call receives 500 submissions from over 50 countries across six continents.

Insights from Climate Policy: Engaging Subnational Governments in Global Platforms 10 June 2020 — 2:45PM TO 6:00PM Anonymous (not verified) 9 February 2021 Online

How have subnational governments shaped the global agenda and created momentum on climate change where national and international governance processes could not?

Can these advances be converted into meaningful collaboration channels for policy development? What works, or does not, when it comes to engagement with multilateral negotiation processes? What ingredients are necessary for success? What are the broader implications of these trends for inclusivity and innovation in international governance?

This event is part of the Inclusive Governance Initiative, which is examining how to build more inclusive models and mechanisms of global governance fit for purpose in today’s world.

Undercurrents: The Oversight Board's Trump decision, and Merkel's legacy Audio bhorton.drupal 25 June 2021

Was Facebook right to suspend Trump? And how will Merkel be remembered?

In the wake of the storming of Capitol Hill on 6 January 2021, social media platforms took steps to remove former President Donald Trump from their websites for infringing community standards. This step was welcomed by many, but also raised serious questions about the power of social media companies to limit free speech and censor elected officials. The suspension of President Trump from Facebook was referred to the Oversight Board, an independent body of experts set up to scrutinise the platform’s content moderation decisions.  

In this episode, Ben speaks to Thomas Hughes and Kate Jones about the outcome of the Oversight Board’s inquiry into the Trump suspension, and the wider implications for content moderation on social media.  

Then Lara is joined by Hans Kundnani to assess the political outlook in Germany and reflect on the legacy of outgoing Chancellor Angela Merkel.  

CARM1 is a protein arginine methyltransferase (PRMT) that acts as a coactivator in a number of transcriptional programs. CARM1 orchestrates this coactivator activity in part by depositing the H3R17me2a histone mark in the vicinity of gene promoters that it regulates. However, the gross levels of H3R17me2a in CARM1 KO mice did not significantly decrease, indicating that other PRMT(s) may compensate for this loss. We thus performed a screen of type I PRMTs, which revealed that PRMT6 can also deposit the H3R17me2a mark in vitro. CARM1 knockout mice are perinatally lethal and display a reduced fetal size, whereas PRMT6 null mice are viable, which permits the generation of double knockouts. Embryos that are null for both CARM1 and PRMT6 are noticeably smaller than CARM1 null embryos, providing in vivo evidence of redundancy. Mouse embryonic fibroblasts (MEFs) from the double knockout embryos display an absence of the H3R17me2a mark during mitosis and increased signs of DNA damage. Moreover, using the combination of CARM1 and PRMT6 inhibitors suppresses the cell proliferation of WT MEFs, suggesting a synergistic effect between CARM1 and PRMT6 inhibitions. These studies provide direct evidence that PRMT6 also deposits the H3R17me2a mark and acts redundantly with CARM1.

Hepatocyte nuclear factor-1β (HNF-1β) is a tissue-specific transcription factor that is required for normal kidney development and renal epithelial differentiation. Mutations of HNF-1β produce congenital kidney abnormalities and inherited renal tubulopathies. Here, we show that ablation of HNF-1β in mIMCD3 renal epithelial cells results in activation of β-catenin and increased expression of lymphoid enhancer–binding factor 1 (LEF1), a downstream effector in the canonical Wnt signaling pathway. Increased expression and nuclear localization of LEF1 are also observed in cystic kidneys from Hnf1b mutant mice. Expression of dominant-negative mutant HNF-1β in mIMCD3 cells produces hyperresponsiveness to exogenous Wnt ligands, which is inhibited by siRNA-mediated knockdown of Lef1. WT HNF-1β binds to two evolutionarily conserved sites located 94 and 30 kb from the mouse Lef1 promoter. Ablation of HNF-1β decreases H3K27 trimethylation repressive marks and increases β-catenin occupancy at a site 4 kb upstream to Lef1. Mechanistically, WT HNF-1β recruits the polycomb-repressive complex 2 that catalyzes H3K27 trimethylation. Deletion of the β-catenin–binding domain of LEF1 in HNF-1β–deficient cells abolishes the increase in Lef1 transcription and decreases the expression of downstream Wnt target genes. The canonical Wnt target gene, Axin2, is also a direct transcriptional target of HNF-1β through binding to negative regulatory elements in the gene promoter. These findings demonstrate that HNF-1β regulates canonical Wnt target genes through long-range effects on histone methylation at Wnt enhancers and reveal a new mode of active transcriptional repression by HNF-1β.

Expert

The functional mechanisms of multidomain proteins often exploit interdomain interactions, or “cross-talk.” An example is human Pin1, an essential mitotic regulator consisting of a Trp–Trp (WW) domain flexibly tethered to a peptidyl-prolyl isomerase (PPIase) domain, resulting in interdomain interactions important for Pin1 function. Substrate binding to the WW domain alters its transient contacts with the PPIase domain via means that are only partially understood. Accordingly, we have investigated Pin1 interdomain interactions using NMR paramagnetic relaxation enhancement (PRE) and molecular dynamics (MD) simulations. The PREs show that apo-Pin1 samples interdomain contacts beyond the range suggested by previous structural studies. They further show that substrate binding to the WW domain simultaneously alters interdomain separation and the internal conformation of the WW domain. A 4.5-μs all-atom MD simulation of apo-Pin1 suggests that the fluctuations of interdomain distances are correlated with fluctuations of WW domain interresidue contacts involved in substrate binding. Thus, the interdomain/WW domain conformations sampled by apo-Pin1 may already include a range of conformations appropriate for binding Pin1's numerous substrates. The proposed coupling between intra-/interdomain conformational fluctuations is a consequence of the dynamic modular architecture of Pin1. Such modular architecture is common among cell-cycle proteins; thus, the WW–PPIase domain cross-talk mechanisms of Pin1 may be relevant for their mechanisms as well.

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6 to 22 μm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.

A. V. Domrina
Trans. Moscow Math. Soc. 83 (), 201-215.
Abstract, references and article information