A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

Molecular identification of peptidoglycan recognition protein 5 and its functional characterization in innate immunity of large yellow croaker, Larimichthys crocea.

Fish peptidoglycan recognition proteins (PGRPs) play important roles in microbial recognition, and bacterial elimination. In the present study, a short-type PGRP from large yellow croaker, LcPGRP5 was cloned and its functions were characterized. LcPGRP5 gene encodes a protein containing conserved PGRP domain, but no signal peptide. Phylogenetic analysis shows that LcPGRP5 is clustered with other short PGRPs identified in other teleosts. LcPGRP5 is constitutively expressed in all tissues examined, with the highest expression being detected in the head kidney. Recombinant LcPGRP5 protein features amidase activity and bactericidal activity. Notably, LcPGRP5 could enhance the phagocytosis of the bacteria by large yellow croaker macrophage, with higher phagocytic capacity being observed in Staphylococcus aureus compared to Escherichia coli. Moreover, overexpression of LcPGRP5 suppresses pro-inflammatory effects elicited by bacterial exposure in the macrophage cell line. Overall, the present results clearly indicate the important roles of LcPGRP5 played in the innate immune responses against bacterial infection.

Purification, secondary structure and antioxidant activity of metallothionein zinc-binding proteins from Arca subcrenata.

Zinc-binding proteins named MT-M-I and MT-M-II were obtained after purification from metal-exposed hairy clams (Arca subcrenata) using gel permeation and ion-exchange chromatography. MT-M-I and MT-M-II were resolved by ion-exchange chromatography, and they were found to have similar molecular weights. MT-M-I and MT-M-II can bind 6 and 7 equivalents of Zn2+ in vitro, and they showed unusual migration behaviors in Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Such migration behaviors may be due to themetal thiolate clusters in these proteins. In terms of amino acid composition, the proportion of cysteine in MT-M-I and MT-M-II was approximately 30%, and glycine accounted for approximately 15%, where as aromatic amino acids were absent. Considering the performance in Tricine-SDS-PAGE and the amino acid compositions, MT-M-I and MT-M-II conform to the molecular characteristics of the metallothionein proteins. The structures were explored using circular dichroism (CD) and Fourier-transform infrared spectroscopy (FTIR). Also determined the antioxidant activities in terms of DPPH radical scavenging ability, hydroxyl radical (·OH) scavenging ability, and ferric-reducing/antioxidant power. The antioxidant activities of MT-M-I were found to be stronger than those of MT-M-II.

Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins.

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

Purification and Assays of Tachycitin.

An antimicrobial peptide tachycitin (73 amino acids) is purified by steps of chromatography, including Sephadex G-50 and S Sepharose FF, from the acid extract of hemocyte debris of horseshoe crabs. Tachycitin is present in monomer form in solution, revealed by ultracentrifugation analysis. Tachycitin exhibits bacterial agglutination activity and inhibits the growth of both Gram-negative bacteria, Gram-positive bacteria, and fungus Candida albicans. Interestingly, tachycitin shows synergistic antimicrobial activity in corporation with another antimicrobial peptide, big defensin. Tachycitin shows a specific binding activity to chitin but not to cellulose, mannan, xylan, and laminarin. Tachycitin is composed of the N-terminal three-stranded ß-sheet and the C-terminal two-stranded ß-sheet following a short helical turn, and the C-terminal structural motif shares a significant structural similarity with the chitin-binding domain derived from a plant chitin-binding protein, hevein.

FMNL2 regulates dynamics of fascin in filopodia.

Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.

Expression and purification of the heme exporter FLVCR1a.

With many crucial roles in enzymatic aerobic metabolism, the concentration of the heme must be tightly regulated. The heme exporter Feline Leukemia Virus sub-group C Receptor 1a (FLVCR1a), an integral membrane protein with twelve transmembrane helices, is a key player in the maintenance of cellular heme homeostasis. It was first identified as the host receptor for the Feline Leukemia Virus sub-group C (FeLV-C), a retrovirus causing hematological abnormalities in cats and other felines. Mutations in the Flvcr1 were later identified in human patients affected by Posterior Column Ataxia and Retinitis Pigmentosa (PCARP) and Hereditary Sensory and Autonomic Neuropathies (HSANs). Despite being an essential component in heme balance, currently there is a lack in the understanding of its function at the molecular level, including the effect of disease-causing mutations on protein function and structure. Therefore, there is a need for protocols to achieve efficient recombinant production yielding milligram amounts of highly pure protein to be used for biochemical and structural studies. Here, we report the first FLVCR1a reliable protocol suitable for both antibody generation and structural characterisation.

Ps19, a novel chitin binding protein from Pteria sterna capable to mineralize aragonite plates in vitro.

Mollusk shell is composed of two CaCO3 polymorphs (calcite and aragonite) and an organic matrix that consists of acetic acid- or ethylenediaminetetraacetic acid (EDTA)-soluble and insoluble proteins and other biomolecules (polysaccharides, ß-chitin). However, the shell matrix proteins involved in nacre formation are not fully known. Thus, the aim of this study was to identify and characterize a novel protein from the acetic acid-insoluble fraction from the shell of Pteria sterna, named in this study as Ps19, to have a better understanding of the biomineralization process. Ps19 biochemical characterization showed that it is a glycoprotein that exhibits calcium- and chitin-binding capabilities. Additionally, it is capable of inducing aragonite plate crystallization in vitro. Ps19 partial peptide sequence showed similarity with other known shell matrix proteins, but it displayed similarity with proteins from Crassostrea gigas, Mizuhopecten yessoensis, Biomphalaria glabrata, Alpysia californica, Lottia gigantea and Elysia chlorotica. The results obtained indicated that Ps19 might play an important role in nacre growth of mollusk shells.

Identification of plasma binding proteins for glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K cells, has potent insulin-releasing and extrapancreatic glucoregulatory activities. However, exogenous GIP has less potent biological effects compared with another incretin hormone, GLP-1, which limits its use for the treatment of type 2 diabetes. The fate and secretion of administered native GIP remain unclear. The aim of this study was to identify plasma binding proteins for human GIP. Fluorescent-labelled GIP was added to fresh human plasma and subjected to clear native polyacrylamide gel electrophoresis (CN-PAGE). Then fluorescent protein bands were in-gel trypsin-digested and subjected to liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, revealing the presence of albumin, immunoglobulin G (IgG) and transferrin. In contrast to GIP, the binding of fluorescent GLP-1 and glucagon to plasma protein fractions were minimal. CN-PAGE analysis of synthetic GIP incubated with human serum albumin, purified IgG or transferrin, and subsequent western blot analysis revealed that GIP binds to each of these proteins. Taken together, these results indicate that GIP readily binds to albumin, IgG and transferrin, three plasma proteins highly abundant in the human peripheral circulation. Separation of protein complexes using CN-PAGE and the identification of in-gel digested proteins by LC-MS/MS analysis provide a promising strategy to identify plasma binding proteins for bioactive peptides.

First Crystal Structure of a Nonstructural Hepatitis E Viral Protein Identifies a Putative Novel Zinc-Binding Protein.

Hepatitis E virus (HEV) is a 7.2-kb positive-sense, single-stranded RNA virus containing three partially overlapping reading frames, ORF1 to ORF3. All nonstructural proteins required for viral replication are encoded by ORF1 and are transcribed as a single transcript. Computational analysis of the complete ORF1 polyprotein identified a previously uncharacterized region of predicted secondary structure bordered by two disordered regions coinciding partially with a region predicted as a putative cysteine protease. Following successful cloning, expression, and purification of this region, the crystal structure of the identified protein was determined and identified to have considerable structural homology to a fatty acid binding domain. Further analysis of the structure revealed a metal binding site, shown unambiguously to specifically bind zinc via a nonclassical, potentially catalytic zinc-binding motif. Based on the structural homology of the HEV protein with known structures, along with the presence of a catalytic zinc-binding motif, it is possible that the identified protein corresponds to the HEV protease, which could require activation or repression through the binding of a fatty acid. This represents a significant step forward in the characterization and the understanding of the molecular mechanisms of the HEV genome. We present analysis for the first time of this identified nonstructural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology.IMPORTANCE Hepatitis E virus (HEV) is an emerging virus found predominately in developing countries; it causes an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the nonstructural proteins of HEV ORF1 are expressed as a single transcript, there is debate as to whether ORF1 functions as a single polyprotein or if it is processed into separate domains via a viral or endogenous cellular protease. Here we present the first structural and biophysical characterization of an HEV nonstructural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease.

Preclinical Development and First-in-Human Imaging of the Integrin αvß6 with [18F]αvß6-Binding Peptide in Metastatic Carcinoma.

PURPOSE: The study was undertaken to develop and evaluate the potential of an integrin αvß6-binding peptide (αvß6-BP) for noninvasive imaging of a diverse range of malignancies with PET. EXPERIMENTAL DESIGN: The peptide αvß6-BP was prepared on solid phase and radiolabeled with 4-[18F]fluorobenzoic acid. In vitro testing included ELISA, serum stability, and cell binding studies using paired αvß6-expressing and αvß6-null cell lines. In vivo evaluation (PET/CT, biodistribution, and autoradiography) was performed in a mouse model bearing the same paired αvß6-expressing and αvß6-null cell xenografts. A first-in-human PET/CT imaging study was performed in patients with metastatic lung, colon, breast, or pancreatic cancer. RESULTS: [18F]αvß6-BP displayed excellent affinity and selectivity for the integrin αvß6 in vitro [IC50(αvß6) = 1.2 nmol/L vs IC50(αvß3) >10 µmol/L] in addition to rapid target-specific cell binding and internalization (72.5% ± 0.9% binding and 52.5% ± 1.8%, respectively). Favorable tumor affinity and selectivity were retained in the mouse model and excretion of unbound [18F]αvß6-BP was rapid, primarily via the kidneys. In patients, [18F]αvß6-BP was well tolerated without noticeable adverse side effects. PET images showed significant uptake of [18F]αvß6-BP in both the primary lesion and metastases, including metastasis to brain, bone, liver, and lung. CONCLUSIONS: The clinical impact of [18F]αvß6-BP PET imaging demonstrated in this first-in-human study is immediate for a broad spectrum of malignancies.

Proteomic differences between focal and diffuse traumatic brain injury in human brain tissue.

The early molecular response to severe traumatic brain injury (TBI) was evaluated using biopsies of structurally normal-appearing cortex, obtained at location for intracranial pressure (ICP) monitoring, from 16 severe TBI patients. Mass spectrometry (MS; label free and stable isotope dimethyl labeling) quantitation proteomics showed a strikingly different molecular pattern in TBI in comparison to cortical biopsies from 11 idiopathic normal pressure hydrocephalus patients. Diffuse TBI showed increased expression of peptides related to neurodegeneration (Tau and Fascin, p < 0.05), reduced expression related to antioxidant defense (Glutathione S-transferase Mu 3, Peroxiredoxin-6, Thioredoxin-dependent peroxide reductase; p < 0.05) and increased expression of potential biomarkers (e.g. Neurogranin, Fatty acid-binding protein, heart p < 0.05) compared to focal TBI. Proteomics of human brain biopsies displayed considerable molecular heterogeneity among the different TBI subtypes with consequences for the pathophysiology and development of targeted treatments for TBI.

Isolation and Characterization of Wheat Derived Nonspecific Lipid Transfer Protein 2 (nsLTP2).

Numerous studies support the protective role of bioactive peptides against cardiovascular diseases. Cereals represent the primary source of carbohydrates, but they also contain substantial amounts of proteins, therefore representing a potential dietary source of bioactive peptides with nutraceutical activities. The analysis of wheat extracts purified by chromatographic techniques by means of HPLC-UV/nanoLC-nanoESI-QTOF allowed the identification of a signal of about 7 kDa which, following data base searches, was ascribed to a nonspecific lipid-transfer protein (nsLTP) type 2 from Triticum aestivum (sequence coverage of 92%). For the first time nsLTP2 biological activities have been investigated. In particular, in experiments with human umbilical vein endothelial cells (HUVEC), nsLTP2 displayed antioxidant and cytoprotective activities, being able to significantly decrease reactive oxygen species (ROS) levels and to reduce lactate dehydrogenase (LDH) release, generated following oxidative (hydrogen peroxide) and inflammatory (tumor necrosis factor α, interleukin-1ß, and lipopolysaccharide) stimulation. The obtained promising results suggest potential protective role of nsLTP2 in vascular diseases prevention. PRACTICAL APPLICATION: nsLTP 2 peptide is resistant to proteases throughout the gastrointestinal tract and exerts antioxidant and cytoprotective activities. These characteristics could be exploited in vascular diseases prevention.

Identification of oligopeptide-binding protein (OppA) and its role in the virulence of Streptococcus suis serotype 2.

The oligopeptide permease (Opp) cassette, an oligopeptide transport system belongs to the superfamily of ATP-binding cassette (ABC) transporter, is widely distributed in bacteria, including Streptococcus suis (S. suis). It is encoded by the opp operon containing oppA, oppB, oppC, oppD, and oppF. In addition to the uptake of peptide, the oppA gene also plays an important role in virulence of many pathogens. In this study, an oppA homologue from the highly virulent S. suis serotype 2 (S. suis 2) strain 05ZYH33 was identified. Flow cytometry and Western blot confirmed that OppA is a surface immunogenic protein and is expressed during S. suis 2 infection. To explore the role of oppA in S. suis 2 growth and pathogenicity, an isogenic 05ZYH33 mutant of oppA (△oppA) was obtained by homologous recombination. Although the complementary strain was not obtained due to the △oppA strain is not transformable, the current data revealed that deletion of the oppA gene in S. suis 2 has greatly affected its growth and virulence. Our data revealed that the growth rate is significantly slow for the △oppA. Adherence of the △oppA strain to human epithelial cells is greatly reduced comparing to the wild strain. Mouse infection experiment showed that inactivation of oppA greatly attenuated the high pathogenicity of S. suis 2. The observed results suggest that OppA is a surface-exposed protein and plays important roles in the growth and pathogenicity of S. suis 2.

Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP).

BACKGROUND: The microsporidian Enterocytozoon hepatopenaei (EHP) is a spore-forming, intracellular parasite that causes an economically debilitating disease (hepatopancreatic microsporidiosis or HPM) in cultured shrimp. HPM is characterized by growth retardation and wide size variation that can result in economic loss for shrimp farmers. Currently, the infection mechanism of EHP in shrimp is poorly understood, especially at the level of host-parasite interaction. In other microsporidia, spore wall proteins have been reported to be involved in host cell recognition. For the host, heparin, a glycosaminoglycan (GAG) molecule found on cell surfaces, has been shown to be recognized by many parasites such as Plasmodium spp. and Leishmania spp. RESULTS: We identified and characterized the first spore wall protein of EHP (EhSWP1). EhSWP1 contains three heparin binding motifs (HBMs) at its N-terminus and a Bin-amphiphysin-Rvs-2 (BAR2) domain at its C-terminus. A phylogenetic analysis revealed that EhSWP1 is similar to an uncharacterized spore wall protein from Enterospora canceri. In a cohabitation bioassay using EHP-infected shrimp with naïve shrimp, the expression of EhSWP1 was detected by RT-PCR in the naïve test shrimp at 20 days after the start of cohabitation. Immunofluorescence analysis confirmed that EhSWP1 was localized in the walls of purified, mature spores. Subcellular localization by an immunoelectron assay revealed that EhSWP1 was distributed in both the endospore and exospore layers. An in vitro binding assay, a competition assay and mutagenesis studies revealed that EhSWP1 is a bona fide heparin binding protein. CONCLUSIONS: Based on our results, we hypothesize that EhSWP1 is an important host-parasite interaction protein involved in tethering spores to host-cell-surface heparin during the process of infection.

The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action.

Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1R1074H and PHF5AY36C The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5AR38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.

Methods to Study DNA End Resection II: Biochemical Reconstitution Assays.

DNA end resection initiates the largely accurate repair of DNA double-strand breaks (DSBs) by homologous recombination. Specifically, recombination requires the formation of 3' overhangs at DSB sites, which is carried out by nucleases that specifically degrade 5'-terminated DNA. In most cases, DNA end resection is a two-step process, comprising of initial short-range followed by more processive long-range resection. In this chapter, we describe selected assays that reconstitute both the short- and long-range pathways. First, we define methods to study the exonuclease and endonuclease activities of the MRE11-RAD50-NBS1 (MRN) complex in conjunction with phosphorylated cofactor CtIP. This reaction is particularly important to initiate processing of DNA breaks and to recruit components belonging to the subsequent long-range pathway. Next, we describe assays that reconstitute the concerted reactions of Bloom (BLM) or Werner (WRN) helicases that function together with the DNA2 nuclease-helicase, and which are as a complex capable to resect DNA of kilobases in length. The reconstituted reactions allow us to understand how the resection pathways function at the molecular level. The assays will be invaluable to define regulatory mechanisms and to identify inhibitory compounds, which may be valuable in cancer therapy.

Cloning and analysis of peptidoglycan recognition protein-LC and immune deficiency from the diamondback moth, Plutella xylostella.

Peptidoglycan (PGN) exists in both Gram-negative and Gram-positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP-LC and IMD (PxPGRP-LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP-LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP-LC and IMD homologs. PxPGRP-LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP-LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full-length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP-LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella.

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori.

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern-recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N-acetylmuramoyl-L-alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP-S4, a short-form PGRP from the domesticated silkworm, Bombyx mori. The PGRP-S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP-S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP-S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn2+ . Scanning electron microscopy showed that PGRP-S4 disrupted the bacterial cell surface. PGRP-S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP-S4 has multiple functions in immunity.

Semi-Quantitative Mass Spectrometry in AML Cells Identifies New Non-Genomic Targets of the EZH2 Methyltransferase.

Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML.