Towards engineering hormone-binding globulins as drug delivery agents.

The treatment of many diseases such as cancer requires the use of drugs that can cause severe side effects. Off-target toxicity can often be reduced simply by directing the drugs specifically to sites of diseases. Amidst increasingly sophisticated methods of targeted drug delivery, we observed that Nature has already evolved elegant means of sending biological molecules to where they are needed. One such example is corticosteroid binding globulin (CBG), the major carrier of the anti-inflammatory hormone, cortisol. Targeted release of cortisol is triggered by cleavage of CBG's reactive centre loop by elastase, a protease released by neutrophils in inflamed tissues. This work aimed to establish the feasibility of exploiting this mechanism to carry therapeutic agents to defined locations. The reactive centre loop of CBG was altered with site-directed mutagenesis to favour cleavage by other proteases, to alter the sites at which it would release its cargo. Mutagenesis succeeded in making CBG a substrate for either prostate specific antigen (PSA), a prostate-specific serine protease, or thrombin, a key protease in the blood coagulation cascade. PSA is conspicuously overproduced in prostatic hyperplasia and is, therefore, a good way of targeting hyperplastic prostate tissues. Thrombin is released during clotting and consequently is ideal for conferring specificity to thrombotic sites. Using fluorescence-based titration assays, we also showed that CBG can be engineered to bind a new compound, thyroxine-6-carboxyfluorescein, instead of its physiological ligand, cortisol, thereby demonstrating that it is possible to tailor the hormone binding site to deliver a therapeutic drug. In addition, we proved that the efficiency with which CBG releases bound ligand can be increased by introducing some well-placed mutations. This proof-of-concept study has raised the prospect of a novel means of targeted drug delivery, using the serpin conformational change to combat the problem of off-target effects in the treatment of diseases.The research was funded by the Wellcome Trust (http://www.wellcome.ac.uk/) grant no. 082961/Z/07/Z to RJR and was facilitated by a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research. WLC was supported by the Singapore government’s Agency for Science, Technology and Research (http://www.astar.edu.sg/). AZ was supported by a Senior Research Fellowship from the British Heart Foundation (http://www.bhf.org.uk). RJR is supported by a Principal Research Fellowship from the Wellcome Trust.This is the final published version. It originally appeared in PLOS ONE at http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0113402

The fast light of CsI(Na) crystals

The responds of different common alkali halide crystals to alpha-rays and gamma-rays are tested in our research. It is found that only CsI(Na) crystals have significantly different waveforms between alpha and gamma scintillations, while others have not this phenomena. It is suggested that the fast light of CsI(Na) crystals arises from the recombination of free electrons with self-trapped holes of the host crystal CsI. Self-absorption limits the emission of fast light of CsI(Tl) and NaI(Tl) crystals.Comment: 5 pages, 11 figures Submit to Chinese Physics

A Sparse Representation Speech Denoising Method Based on Adapted Stopping Residue Error

A sparse representation speech denoising method based on adapted stopping residue error was presented in this paper. Firstly, the cross-correlation between the clean speech spectrum and the noise spectrum was analyzed, and an estimation method was proposed. In the denoising method, an over-complete dictionary of the clean speech power spectrum was learned with the K-singular value decomposition (K-SVD) algorithm. In the sparse representation stage, the stopping residue error was adaptively achieved according to the estimated cross-correlation and the adjusted noise spectrum, and the orthogonal matching pursuit (OMP) approach was applied to reconstruct the clean speech spectrum from the noisy speech. Finally, the clean speech was re-synthesised via the inverse Fourier transform with the reconstructed speech spectrum and the noisy speech phase. The experiment results show that the proposed method outperforms the conventional methods in terms of subjective and objective measure

Mechanistic studies on regulation of the activity of GPI-anchored serine protease testisin

Objective·To explore the activation methods and activity regulation mechanisms of the testisin zymogen in vitro, laying a foundation for further research on the physiological functions of testisin in organisms.Methods·The eukaryotic expression plasmid for the mouse-derived testisin (mTN) zymogen was constructed through full-gene synthesis and subsequently transfected into eukaryotic HEK293S cells for expression. Purified recombinant testisin protein was obtained through methods such as nickel ion affinity chromatography. Site-directed mutagenesis was performed on the activation site and active center of the zymogen, and the activation mechanism of testisin was analyzed through enzymatic activity assays. Additionally, the activation rate of the protein was determined by altering the incubation conditions of the zymogen. The self-cleavage site of the protease was identified by using Edman degradation and site-directed mutagenesis. Finally, the regulatory mechanism of protease activity was examined by analyzing the proteolytic rate of the protein towards its substrates.Results·The recombinant mTN zymogen was expressed in eukaryotic HEK293S cells, and high-purity, uniform recombinant protein was obtained through a two-step purification process. This protein was found to remain stable under acidic conditions but underwent self-activation in neutral or alkaline environments. The rate of activation was influenced by factors such as pH value and temperature. The self-activation process required on the integrity of the enzyme's activation site, Arg46, and its active center, Ser240. Additionally, the self-activation of the recombinant mTN zymogen was often accompanied by the self-cleavage at the 170/175 loop on the molecular surface, but this self-cleavage did not affect the activity of testisin. Furthermore, the optimal reaction pH for activated mTN was 8.0, with the protein remaining relatively stable near this pH. The optimal reaction temperature was 50 ℃, while the protein was best stored at temperatures below 30 ℃. Zn2+ and Ca2+ were found to significantly inhibit mTN activity.Conclusion·An effective method for the preparation of recombinant mTN zymogen is successfully established, and it is discovered that the recombinant mTN undergoes self-activation and self-cleavage in vitro

Structural basis for the specificity of renin-mediated angiotensinogen cleavage.

The renin-angiotensin cascade is a hormone system that regulates blood pressure and fluid balance. Renin-mediated cleavage of the angiotensin I peptide from the N terminus of angiotensinogen (AGT) is the rate-limiting step of this cascade; however, the detailed molecular mechanism underlying this step is unclear. Here, we solved the crystal structures of glycosylated human AGT (2.30 Å resolution), its encounter complex with renin (2.55 Å), AGT cleaved in its reactive center loop (RCL; 2.97 Å), and spent AGT from which the N-terminal angiotensin peptide was removed (2.63 Å). These structures revealed that AGT undergoes profound conformational changes and binds renin through a tail-into-mouth allosteric mechanism that inserts the N terminus into a pocket equivalent to a hormone-binding site on other serpins. These changes fully extended the N-terminal tail, with the scissile bond for angiotensin release docked in renin's active site. Insertion of the N terminus into this pocket accompanied a complete unwinding of helix H of AGT, which, in turn, formed key interactions with renin in the complementary binding interface. Mutagenesis and kinetic analyses confirmed that renin-mediated production of angiotensin I is controlled by interactions of amino acid residues and glycan components outside renin's active-site cleft. Our findings indicate that AGT adapts unique serpin features for hormone delivery and binds renin through concerted movements in the N-terminal tail and in its main body to modulate angiotensin release. These insights provide a structural basis for the development of agents that attenuate angiotensin release by targeting AGT's hormone binding pocket

Preparation and target protease identification of a cyanobacterial serine protease inhibitor, arthropin

Objective·To prepare a high-purity cyanobacterial serine protease inhibitor, screen its target proteases, and detect its inhibitory activity.Methods·A novel serine protease inhibitor from Arthrospira platensis was identified in the Expanded Human Oral Microbiome Database (eHOMD) by amino acid sequence alignment and named as arthropin. The fusion expression vector pSUMO3-arthropin was constructed and transferred into Escherichia coli (E. coli) BL21(DE3) system for fusion protein expression. The recombinant arthropin was purified by a four-step chromatographic purification approach of nickel affinity chromatography, enzymatic digestion, reverse nickel affinity chromatography, and anion exchange chromatography. In addition, the recombinant arthropin was co-incubated with 14 serine proteases such as activated factor Ⅸ (FⅨa), FⅩa, FⅪa, activated protein C (APC) and kallikrein 1 (KLK1), respectively, and then analyzed by SDS-PAGE. The inhibitory rate of arthropin on KLK1 was assayed with kinetic methods. The crystallization conditions of the recombinant arthropin were screened preliminarily, and the suitable crystals were picked for X-ray diffraction to collect the data. Finally, a sub-stable structure model of arthropin was predicted with AlphaFlod Colab.Results·SDS-PAGE showed that the fused arthropin was successfully expressed in the E. coli BL21(DE3) system, and following purification, the high-purity recombinant arthropin, the relative molecular mass of which was similar to the theoretical value (45 800), was obtained. The co-incubation analysis of recombinant arthropin with 14 serine proteases revealed that arthropin was able to form stable covalent complexes with 9 proteases, including FⅩa, APC, FⅨa, FⅪa, trypsin, cathepsin G, KLK1, KLK7 and thrombin. Arthropin inhibited KLK1 with a second-order association rate constant of 1.7×103 L/(mol·s). Moreover, the recombinant arthropin crystalised under the condition of 25% PEG MME 550, 0.1 mol/L MES (pH 6.5) and 0.01 mol/L ZnCl2 , and the crystals preliminarily diffracted to a resolution of 10 Å (1 Å=1×10-10 m). The analysis of the structure predicted by AlphaFlod Colab revealed that arthropin had the classical structural features of the inhibitory serpin.Conclusion·Arthropin, a serpin from Arthrospira platensis, was successfully obtained with high purity and a broad-spectrum of serine protease inhibition, but at a low inhibitory rate

Inhibition of Tannerpin-M encoded by periodontal pathogens on serine proteases released by granulocytes

Objective·To prepare a serine protease inhibitor (Serpin) derived from Tannerella which is associated with periodontosis, and analyze its specificity in inhibiting target proteases and its structural characteristics.Methods·Through amino acid sequence analysis, a Serpin from the human oral microbiome database (eHOMD) was selected and expressed in Escherichia coli. The recombinant protein was purified using methods such as nickel ion affinity chromatography. Its specificity in inhibiting serine proteases was analyzed, followed by an analysis of its three-dimensional spatial structure using structural biology methods.Results·A novel Serpin, named Tannerpin-M, with methionine as the active center P1 residue, was identified, and a high-purity recombinant protein was successfully prepared from Escherichia coli BL21 (DE3). Further activity testing demonstrated that recombinant Tannerpin-M could effectively form SDS-stable covalent complexes with proteases derived from granulocytes (human neutrophil elastase, cathepsin G, and proteinase 3), as well as with other proteases including kallikrein 1 (KLK1), KLK7, and elastase. Tannerpin-M inhibited KLK7 with a second-order association rate constant of 4.12×104 L/(mol·s). The crystal structure of Tannerpin-M in its relaxed state conformation was resolved at a resolution of 2.4 Å (1 Å=0.1 nm). It revealed that Tannerpin-M possessed a significantly elongated reactive center loop and could undergo the classical conformational transition from a stressed to a relaxed state.Conclusion·Tannerpin-M, derived from oral pathogenic bacteria, is a typical inhibitory Serpin, and can effectively inhibit the serine protease released by granulocytes, by which it may protect the oral pathogenic bacteria from attacks of the human immune system

Temperature-responsive release of thyroxine and its environmental adaptation in Australians.

The hormone thyroxine that regulates mammalian metabolism is carried and stored in the blood by thyroxine-binding globulin (TBG). We demonstrate here that the release of thyroxine from TBG occurs by a temperature-sensitive mechanism and show how this will provide a homoeostatic adjustment of the concentration of thyroxine to match metabolic needs, as with the hypothermia and torpor of small animals. In humans, a rise in temperature, as in infections, will trigger an accelerated release of thyroxine, resulting in a predictable 23% increase in the concentration of free thyroxine at 39°C. The in vivo relevance of this fever-response is affirmed in an environmental adaptation in aboriginal Australians. We show how two mutations incorporated in their TBG interact in a way that will halve the surge in thyroxine release, and hence the boost in metabolic rate that would otherwise occur as body temperatures exceed 37°C. The overall findings open insights into physiological changes that accompany variations in body temperature, as notably in fevers

NAP1 Family Histone Chaperones Are Required for Somatic Homologous Recombination in <i>Arabidopsis</i>

Homologous recombination (HR) is essential for maintaining genome integrity and variability. To orchestrate HR in the context of chromatin is a challenge, both in terms of DNA accessibility and restoration of chromatin organization after DNA repair. Histone chaperones function in nucleosome assembly/disassembly and could play a role in HR. Here, we show that the NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) family histone chaperones are required for somatic HR in Arabidopsis thaliana. Depletion of either the NAP1 group or NAP1-RELATED PROTEIN (NRP) group proteins caused a reduction in HR in plants under normal growth conditions as well as under a wide range of genotoxic or abiotic stresses. This contrasts with the hyperrecombinogenic phenotype caused by the depletion of the CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) histone chaperone. Furthermore, we show that the hyperrecombinogenic phenotype caused by CAF-1 depletion relies on NRP1 and NRP2, but the telomere shortening phenotype does not. Our analysis of DNA lesions, H3K56 acetylation, and expression of DNA repair genes argues for a role of NAP1 family histone chaperones in nucleosome disassembly/reassembly during HR. Our study highlights distinct functions for different families of histone chaperones in the maintenance of genome stability and establishes a crucial function for NAP1 family histone chaperones in somatic HR

Linking PHYTOCHROME-INTERACTING FACTOR to Histone Modification in Plant Shade Avoidance

Shade avoidance syndrome (SAS) allows a plant grown in a densely populated environment to maximize opportunities to access to sunlight. Although it is well established that SAS is accompanied by gene expression changes, the underlying molecular mechanism needs to be elucidated. Here, we identify the H3K4me3/H3K36me3-binding proteins, Morf Related Gene (MRG) group proteins MRG1 and MRG2, as positive regulators of shade-induced hypocotyl elongation in Arabidopsis (Arabidopsis thaliana). MRG2 binds PHYTOCHROME-INTERACTING FACTOR7 (PIF7) and regulates the expression of several common downstream target genes, including YUCCA8 and IAA19 involved in the auxin biosynthesis or response pathway and PRE1 involved in brassinosteroid regulation of cell elongation. In response to shade, PIF7 and MRG2 are enriched at the promoter and gene-body regions and are necessary for increase of histone H4 and H3 acetylation to promote target gene expression. Our study uncovers a mechanism in which the shade-responsive factor PIF7 recruits MRG1/MRG2 that binds H3K4me3/H3K36me3 and brings histone-acetylases to induce histone acetylations to promote expression of shade responsive genes, providing thus a molecular mechanistic link coupling the environmental light to epigenetic modification in regulation of hypocotyl elongation in plant SAS