Microsecond folding dynamics of the F13W G29A mutant of the B domain of staphylococcal protein A by laser-induced temperature jump

The small size (58 residues) and simple structure of the B domain of staphylococcal protein A (BdpA) have led to this domain being a paradigm for theoretical studies of folding. Experimental studies of the folding of BdpA have been limited by the rapidity of its folding kinetics. We report the folding kinetics of a fluorescent mutant of BdpA (G29A F13W), named F13W*, using nanosecond laser-induced temperature jump experiments. Automation of the apparatus has permitted large data sets to be acquired that provide excellent signal-to-noise ratio over a wide range of experimental conditions. By measuring the temperature and denaturant dependence of equilibrium and kinetic data for F13W*, we show that thermodynamic modeling of multidimensional equilibrium and kinetic surfaces is a robust method that allows reliable extrapolation of rate constants to regions of the folding landscape not directly accessible experimentally. The results reveal that F13W* is the fastest-folding protein of its size studied to date, with a maximum folding rate constant at 0 M guanidinium chloride and 45°C of 249,000 (s-1). Assuming the single-exponential kinetics represent barrier-limited folding, these data limit the value for the preexponential factor for folding of this protein to at least ≈2 x 10(6) s(-1)

DNA binding redistributes activation domain ensemble and accessibility in pioneer factor Sox2

More than 1600 human transcription factors orchestrate the transcriptional machinery to control gene expression and cell fate. Their function is conveyed through intrinsically disordered regions (IDRs) containing activation or repression domains but lacking quantitative structural ensemble models prevents their mechanistic decoding. Here we integrate single-molecule FRET and NMR spectroscopy with molecular simulations showing that DNA binding can lead to complex changes in the IDR ensemble and accessibility. The C-terminal IDR of pioneer factor Sox2 is highly disordered but its conformational dynamics are guided by weak and dynamic charge interactions with the folded DNA binding domain. Both DNA and nucleosome binding induce major rearrangements in the IDR ensemble without affecting DNA binding affinity. Remarkably, interdomain interactions are redistributed in complex with DNA leading to variable exposure of two activation domains critical for transcription. Charged intramolecular interactions allowing for dynamic redistributions may be common in transcription factors and necessary for sensitive tuning of structural ensembles

A retrospective and agenda for future research on Chinese outward foreign direct investment

Our original paper “The determinants of Chinese Outward Foreign Direct Investment” was the first theoretically based empirical analysis of the phenomenon. It utilised internalisation theory to show that Chinese state-owned firms reacted to home country market imperfections to surmount barriers to foreign entry arising from naivety and the lack of obvious ownership advantages, leveraging institutional factors including favourable policy stimuli. This special theory explained outward foreign direct investment (OFDI) but provided surprises. These included the apparent appetite for risk evinced by these early investors, causing us to conjecture that domestic market imperfections, particularly in the domestic capital market, might be responsible. The article stimulated a massive subsequent, largely successful, research effort on emerging country multinationals. In this Retrospective article we review some of the main strands of research that ensued, for the insight they offer for the theme of our commentary. Our theme is that theoretical development can only come through embracing yet more challenging, different, and new contexts, and we make suggestions for future research directions

Advancing the field of viroporins—Structure, function and pharmacology: IUPHAR Review X

Viroporins possess important potential as antiviral targets due to their critical roles during virus life cycles, spanning from virus entry to egress. Although the antiviral amantadine targets the M2 viroporin of influenza A virus, successful progression of other viroporin inhibitors into clinical use remains challenging. These challenges relate in varying proportions to a lack of reliable full-length 3D-structures, difficulties in functionally characterising individual viroporins, and absence of verifiable direct binding between inhibitor and viroporin. This review offers perspectives to help overcome these challenges. We provide a comprehensive overview of the viroporin family, including their structural and functional features, highlighting the moldability of their energy landscapes and actions. To advance the field, we suggest a list of best practices to aspire towards unambiguous viroporin identification and characterisation, along with considerations of potential pitfalls. Finally, we present current and future scenarios of, and prospects for, viroporin targeting drugs

The Mutational Profile of Unicystic Ameloblastoma

BRAF V600E is the most common mutation in conventional ameloblastoma (AM) of the mandible. In contrast, maxillary AMs appear to harbor more frequently RAS, FGFR2, or SMO mutations. Unicystic ameloblastoma (UAM) is considered a less aggressive variant of ameloblastoma, amenable to more conservative treatment, and classified as a distinct entity. The aim of this study was to characterize the mutation profile of UAM (n = 39) and to compare it to conventional AM (n = 39). The associations between mutation status and recurrence probability were also analyzed. In the mandible, 94% of UAMs (29/31, including 8/8 luminal, 6/8 intraluminal, and 15/15 mural subtypes) and 74% of AMs (28/38) revealed BRAF V600E mutations. Among the BRAF wild-type cases, 1 UAM showed a missense SMO mutation (p.L412F), whereas 2 NRAS (p.Q61R), 2 HRAS (p.Q61R), and 2 FGFR2 (p.C383R) activating mutations were identified in AM. Of the 3 maxillary UAMs, only 1 revealed a BRAF V600E mutation. Taken together, our findings demonstrate high frequency of activating BRAF V600E mutations in both UAM and AM of the mandible. In maxillary UAMs, the BRAF V600E mutation prevalence appears to be lower as was shown for AM previously. It could therefore be argued that UAM and AM are part of the spectrum of the same disease. AMs without BRAF V600E mutations were associated with an increased rate of local recurrence (P = 0.0003), which might indicate that routine mutation testing also has an impact on prognosis.Peer reviewe

Peptides containing the PCNA interacting motif APIM bind to the β-clamp and inhibit bacterial growth and mutagenesis

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents

A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation

Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial–mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer