Uncovering the non-histone interactome of the BRPF1 bromodomain using site-specific azide-acetyllysine photochemistry.

Bromodomain-PHD finger protein 1 (BRPF1) belongs to the BRPF family of bromodomain-containing proteins. Bromodomains are exclusive reader modules that recognize and bind acetylated histones and non-histone transcription factors to regulate gene expression. The biological functions of acetylated histone recognition by BRPF1 bromodomain are well characterized; however, the function of BRPF1 regulation via non-histone acetylation is still unexplored. Therefore, identifying the non-histone interactome of BRPF1 is pivotal in deciphering its role in diverse cellular processes, including its misregulation in diseases like cancer. Herein, we identified the non-histone interacting partners of BRPF1 utilizing a protein engineering-based approach. We site-specifically introduced the unnatural photo-cross-linkable amino acid 4-azido-L-phenylalanine into the bromodomain of BRPF1 without altering its ability to recognize acetylated histone proteins. Upon photoirradiation, the engineered BRPF1 generates a reactive nitrene species, cross-linking interacting partners with spatio-temporal precision. We demonstrated the robust cross-linking efficiency of the engineered variant with reported histone ligands of BRPF1 and further used the variant reader to cross-link its interactome. We also characterized novel interacting partners by proteomics, suggesting roles for BRPF1 in diverse cellular processes. BRPF1 interaction with interleukin enhancer-binding factor 3, one of these novel interacting partners, was further validated by isothermal titration calorimetry and co-IP. Lastly, we used publicly available ChIP-seq and RNA-seq datasets to understand the colocalization of BRPF1 and interleukin enhancer-binding factor 3 in regulating gene expression in the context of hepatocellular carcinoma. Together, these results will be crucial for full understanding of the roles of BRPF1 in transcriptional regulation and in the design of small-molecule inhibitors for cancer treatment.

Amyloid ß-Peptide Segment Conjugated Side-Chain Proline-Based Polymers as Potent Inhibitors in Lysozyme Amyloidosis.

Developing effective amyloidosis inhibitors poses a significant challenge due to the dynamic nature of the protein structures, the complex interplay of interfaces in protein-protein interactions, and the irreversible nature of amyloid assembly. The interactions of amyloidogenic polypeptides with other peptides play a pivotal role in modulating amyloidosis and fibril formation. This study presents a novel approach for designing and synthesizing amyloid interaction surfaces using segments derived from the amyloid-promoting sequence of amyloid ß-peptide [VF(Aß(18-19)/FF(Aß(19-20)/LVF(Aß(17-19)/LVFF(Aß(17-20)], where VF, FF, LVF and LVFF stands for valine phenylalanine dipeptide, phenylalanine phenylalanine dipeptide, leucine valine phenylalanine tripeptide and leucine valine phenylalanine phenylalanine tetrapeptide, respectively. These segments are conjugated with side-chain proline-based methacrylate polymers serving as potent lysozyme amyloidosis inhibitors and demonstrating reduced cytotoxicity of amyloid aggregations. Di-, tri-, and tetra-peptide conjugated chain transfer agents (CTAs) were synthesized and used for the reversible addition-fragmentation chain transfer polymerization of tert-butoxycarbonyl (Boc)-proline methacryloyloxyethyl ester (Boc-Pro-HEMA). Deprotection of Boc-groups from the side-chain proline pendants resulted in water-soluble polymers with defined peptide chain ends as peptide-polymer bioconjugates. Among them, the LVFF-conjugated polymer acted as a potent inhibitor with significantly suppressed lysozyme amyloidosis, a finding supported by comprehensive spectroscopic, microscopic, and computational analyses. These results unveil the synergistic effect between the segment-derived amyloid ß-peptide and side-chain proline-based polymers, offering new prospects for targeting lysozyme amyloidosis.

Novel insights into the recognition of acetylated histone H4 tail by the TRIM24 PHD-Bromo module.

TRIM24 is a multi-functional chromatin reader, and it binds to the estrogen receptor to activate estrogen-dependent target genes associated with tumor development. TRIM24 is known to ubiquitinate p53 via an N-terminal RING domain and binds a specific combinatorial histone signature of H3K4me0/H3K23ac via its C-terminal plant homeodomain (PHD) and bromodomain (Bromo). Aberrant expression of TRIM24 positively correlates with H3K23ac levels, and high levels of both TRIM24 and H3K23ac predict poor survival of breast cancer patients. Little has been explored about the acetylated histone H4 (H4ac) signatures of TRIM24 and their biological functions. Herein, we report novel H4ac binding partners of TRIM24 and their localization in the genome. Isothermal titration calorimetry binding assay on the histone peptides revealed that the TRIM24 PHD-Bromo preferably binds to H4K5ac, H4K8ac, and H4K5acK8ac compared with other acetylated histone H4 ligands. Co-immunoprecipitation on the endogenous histones suggests that the recognition of H4ac by Bromo does not interfere with the recognition of H3K4me0 mark by the PHD domain of TRIM24. Consistent with this, TRIM24 PHD-Bromo exhibits minimal discrimination among H4ac binding partners at endogenous histone and nucleosome levels. Moreover, ChIP-seq analysis revealed that the H4K5ac and H4K8ac histone signatures strongly co-localize near the transcription start sites of different hub genes or TRIM24-targeted genes in breast cancer. In addition, the KEGG pathway analysis demonstrates that the TRIM24 and its H4ac targets are associated with several important biological pathways. Our findings describe that the H4ac recognition by TRIM24 PHD-Bromo enables access to the chromatin for specific transcriptional regulation.

Uncovering the Domain-Specific Interactome of the TAF1 Tandem Reader Using Site-Specific Azide-Acetyllysine Photochemistry.

Combinatorial readout of histone post-translational modifications by tandem reader modules mediates crosstalk among different histone modifications. To identify the domain-specific interactome of the tandem reader, we engineered the dual bromodomain of TATA-binding protein-associated factor-1 (TAF1) to carry a photoactivatable unnatural amino acid, 4-azido-l-phenylalanine (AzF), via amber suppressor mutagenesis. Using computational approaches, we modeled the targeted residues of TAF1 with AzF to predict the cross-linking distance between the reactive arylazide and its interacting partner. We developed three photoactivatable TAF1 tandem-bromodomain analogues, viz., Y1403AzF in bromodomain 1 (BD1), W1526AzF in bromodomain 2 (BD2), and Y1403AzF/W1526AzF in both BD1 and BD2. Circular dichroism and a thermal shift assay were used to confirm the structural integrity of the engineered readers. Using the TAF1 tandem-bromodomain analogues, we characterized their histone ligand binding properties by isothermal titration calorimetry and photo-cross-linking experiments. We found that the dual bromodomain of TAF1 independently binds and cross-links to different acetylated histone ligands. We further used the engineered BD1 and BD2 analogues of the TAF1 tandem readers to identify their domain-specific interacting partners at the cellular level. Both BD1 and BD2 independently cross-link to a unique interactome, and importantly, the dual cross-linker carrying TAF1 analogue could capture both BD1- and BD2-specific interactomes. Our work concludes that BD1 and BD2 of the TAF1 tandem reader independently recognize their interacting partners to regulate downstream cellular functions.

Molecular Insights into the Recognition of Acetylated Histone Modifications by the BRPF2 Bromodomain.

HBO1 [HAT bound to the origin recognition complex (ORC)], a member of the MYST family of histone acetyltransferases (HATs), was initially identified as a binding partner of ORC that acetylates free histone H3, H4, and nucleosomal H3. It functions as a quaternary complex with the BRPF (BRPF1/2/3) scaffolding protein and two accessory proteins, ING4/5 and Eaf6. Interaction of BRPF2 with HBO1 has been shown to be important for regulating H3K14 acetylation during embryonic development. However, how BRPF2 directs the HBO1 HAT complex to chromatin to regulate its HAT activity toward nucleosomal substrates remains unclear. Our findings reveal novel interacting partners of the BRPF2 bromodomain that recognizes different acetyllysine residues on the N-terminus of histone H4, H3, and H2A and preferentially binds to H4K5ac, H4K8ac, and H4K5acK12ac modifications. In addition, mutational analysis of the BRPF2 bromodomain coupled with isothermal titration calorimetry binding and pull-down assays on the histone substrates identified critical residues responsible for acetyllysine binding. Moreover, the BRPF2 bromodomain could enrich H4K5ac mark-bearing mononucleosomes compared to other acetylated H4 marks. Consistent with this, ChIP-seq analysis revealed that BRPF2 strongly co-localizes with HBO1 at histone H4K5ac and H4K8ac marks near the transcription start sites in the genome. Our study provides novel insights into how the histone binding function of the BRPF2 bromodomain directs the recruitment of the HBO1 HAT complex to chromatin to regulate gene expression.

Catalytic Space Engineering as a Strategy to Activate C-H Oxidation on 5-Methylcytosine in Mammalian Genome.

Conditional remodeling of enzyme catalysis is a formidable challenge in protein engineering. Herein, we have undertaken a unique active site engineering tactic to command catalytic outcomes. With ten-eleven translocation (TET) enzyme as a paradigm, we show that variants with an expanded active site significantly enhance multistep C-H oxidation in 5-methylcytosine (5mC), whereas a crowded cavity leads to a single-step catalytic apparatus. We further identify an evolutionarily conserved residue in the TET family with a remarkable catalysis-directing ability. The activating variant demonstrated its prowess to oxidize 5mC in chromosomal DNA for potentiating expression of genes including tumor suppressors.

Complementary Steric Engineering at the Protein-Ligand Interface for Analogue-Sensitive TET Oxygenases.

Ten-eleven translocation (TET) enzymes employ O2, earth-abundant iron, and 2-ketoglutarate (2KG) to perform iterative C-H oxidation of 5-methylcytosine in DNA to control expression of the mammalian genome. Given that more than 60 such C-H oxygenases are present in humans, determining context-dependent functions of each of these enzymes is a pivotal challenge. In an effort to tackle the problem, we developed analogue-sensitive TET enzymes to perturb the activity of a specific member. We rationally engineered the TET2-2KG interface to develop TET2 variants with an expanded active site that can be specifically inhibited by the N-oxalylglycine (NOG) derivatives carrying a complementary steric "bump". Herein, we describe the identification and engineering of a bulky gatekeeper residue for TET proteins, characterize the orthogonal mutant-inhibitor pairs, and show generality of the approach. Employing cell-permeable NOG analogues, we show that the TET2 mutant can be specifically inhibited to conditionally modulate cytosine methylation in chromosomal DNA in intact human cells. Finally, we demonstrate application of the orthogonal mutant-inhibitor pair to probe transcriptional activity of a specific TET member in cells. Our work provides a general platform for developing analogue-sensitive 2KG-dependent oxygenases to unravel their functions in diverse signaling processes.

Specific Acetylation Patterns of H2A.Z Form Transient Interactions with the BPTF Bromodomain.

Post-translational lysine acetylation of histone tails affects both chromatin accessibility and recruitment of multifunctional bromodomain-containing proteins for modulating transcription. The bromodomain- and PHD finger-containing transcription factor (BPTF) regulates transcription but has also been implicated in high gene expression levels in a variety of cancers. In this report, the histone variant H2A.Z, which replaces H2A in chromatin, is evaluated for its affinity for BPTF with a specific recognition pattern of acetylated lysine residues of the N-terminal tail region. Although BPTF immunoprecipitates H2A.Z-containing nucleosomes, a direct interaction with its bromodomain has not been reported. Using protein-observed fluorine nuclear magnetic resonance (PrOF NMR) spectroscopy, we identified a diacetylation of H2A.Z on lysine residues 4 and 11, with the highest affinity for BPTF with a Kd of 780 µM. A combination of subsequent 1H NMR Carr-Purcell-Meiboom-Gill experiments and photo-cross-linking further confirmed the specificity of the diacetylation pattern at lysines 4 and 11. Because of an adjacent PHD domain, this transient interaction may contribute to a higher-affinity bivalent interaction. Further evaluation of specificity toward a set of bromodomains, including two BET bromodomains (Brd4 and BrdT) and two Plasmodium falciparum bromodomains, resulted in one midmicromolar affinity binder, PfGCN5 (Kd = 650 µM). With these biochemical experiments, we have identified a direct interaction of histone H2A.Z with bromodomains with a specific acetylation pattern that further supports the role of H2A.Z in epigenetic regulation.

A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes.

Enzymatic methylation at carbon five on cytosine (5mC) in DNA is a hallmark of mammalian epigenetic programming and is critical to gene regulation during early embryonic development. It has recently been shown that dynamic erasure of 5mC by three members of the ten-eleven translocation (TET) family plays a key role in cellular differentiation. TET enzymes belong to Fe (II)- and 2-ketoglutarate (2KG) dependent dioxygenases that successively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5CaC), thus providing a chemical basis for the removal of 5mC which once was thought to be a permanent mark in mammalian genome. Since then a wide range of biochemical assays have been developed to characterize TET activity. Majority of these methods require multi-step processing to detect and quantify the TET-mediated oxidized products. In this study, we have developed a MALDI mass spectrometry based method that directly measures the TET activity with high sensitivity while eliminating the need for any intermediate processing steps. We applied this method to the measurement of enzymatic activity of TET2 and 3, Michaleis-Menten parameters (KM and kcat) of TET-2KG pairs and inhibitory concentration (IC50) of known small-molecule inhibitors of TETs. We further demonstrated the suitability of the assay to analyze chemoenzymatic labeling of 5hmC by ß-glucosyltransferase, highlighting the potential for broad application of our method in deconvoluting the functions of novel DNA demethylases.

Engineering Biological C-H Functionalization Leads to Allele-Specific Regulation of Histone Demethylases.

Oxidative C-H hydroxylation of methyl groups, followed by their removal from DNA, RNA, or histones, is an epigenetic process critical to transcriptional reprogramming and cell fate determination. This reaction is catalyzed by Fe(II)-dependent dioxygenases using the essential metabolite 2-ketoglutarate (2KG) as a cofactor. Given that the human genome encodes for more than 60 2KG-dependent dioxygenases, assigning their individual functions remains a significant challenge. Here we describe a protein-ligand interface engineering approach to break the biochemical degeneracy of these enzymes. Using histone lysine demethylase 4 as a proof-of-concept, we show that the enzyme active site can be expanded to employ bulky 2KG analogues that do not sensitize wild-type demethylases. We establish the orthogonality, substrate specificity, and catalytic competency of the engineered demethylation apparatus in biochemical assays. We further demonstrate demethylation of cognate substrates in physiologically relevant settings. Our results provide a paradigm for rapid and conditional manipulation of histone demethylases to uncloak their isoform-specific functions.

Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain, and is consistent with Michaelis-Menten mechanism.

BACKGROUND: It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease. METHODS: Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis. RESULTS: The CHS4 domain comprised of sequence residues 116-263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis-Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94-170) within CHS4 is indispensable. CONCLUSION: The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does. SIGNIFICANCE: Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity.

Enzyme dimension of the ribosomal protein S4 across plant and animal kingdoms.

BACKGROUND: The protein S4 of the smaller ribosomal subunit is centrally important for its anchorage role in ribosome assembly and rRNA binding. Eubacterial S4 also facilitates synthesis of rRNA, and restrains translation of ribosomal proteins of the same polycistronic mRNA. Eukaryotic S4 has no homolog in eubacterial kingdom, nor are such extraribosomal functions of S4 known in plants and animals even as genetic evidence suggests that deficiency of S4X isoform in 46,XX human females may produce Turner syndrome (45,XO). METHODS: Recombinant human S4X and rice S4 were used to determine their enzymatic action in the cleavage of synthetic peptide substrates and natural proteins. We also studied autoproteolysis of the recombinant S4 proteins, and examined the growth and proliferation of S4-transfected human embryonic kidney cells. RESULTS: Extraribosomal enzyme nature of eukaryotic S4 is described. Both human S4X and rice S4 are cysteine proteases capable of hydrolyzing a wide spectrum of peptides and natural proteins of diverse origin. Whereas rice S4 also cleaves the -XXXD↓- consensus sequence assumed to be specific for caspase-9 and granzyme B, human S4 does not. Curiously, both human and rice S4 show multiple-site autoproteolysis leading to self-annihilation. Overexpression of human S4 blocks the growth and proliferation of transfected embryonic kidney cells, presumably due to the extraribosomal enzyme trait reported. CONCLUSIONS: The S4 proteins of humans and rice, prototypes of eukaryota, are non-specific cysteine proteases in the extraribosomal milieu. GENERAL SIGNIFICANCE: The enzyme nature of S4 is relevant toward understanding not only the origin of ribosomal proteins, but also processes in cell biology and diseases.

Arrested cell proliferation through cysteine protease activity of eukaryotic ribosomal protein S4.

S4 is an integral protein of the smaller subunit of cytosolic ribosome. In prokaryotes, it regulates the synthesis of ribosomal proteins by feedback inhibition of the α-operon gene expression, and it facilitates ribosomal RNA synthesis by direct binding to RNA polymerase. However, functional roles of S4 in eukaryotes are poorly understood, although its deficiency in humans is thought to produce Turner syndrome. We report here that wheat S4 is a cysteine protease capable of abrogating total protein synthesis in an actively translating cell-free system of rabbit reticulocytes. The translation-blocked medium, imaged by atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, shows dispersed polysomes, and the disbanded polyribosome elements aggregate to form larger bodies. We also show that human embryonic kidney cells transfected with recombinant wheat S4 are unable to grow and proliferate. The mutant S4 protein, where the putative active site residue Cys 41 is replaced by a phenylalanine, can neither suppress protein synthesis nor arrest cell proliferation, suggesting that the observed phenomenon arises from the cysteine protease attribute of S4. The results also inspire many questions concerning in vivo significance of extraribosomal roles of eukaryotic S4 performed through its protease activity.

Integrated virtual screening and MD simulation approaches toward discovering potential inhibitors for targeting BRPF1 bromodomain in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive and life-threatening cancers. Although multiple treatment options are available, the prognosis of HCC patients is poor due to metastasis and drug resistance. Hence, discovering novel targets is essential for better therapeutic development for HCC. In this study, we used the cancer genome atlas (TCGA) dataset to analyze the expression of bromodomain-containing proteins in HCC, as bromodomains are emerging attractive therapeutic targets. Our analysis identified BRPF1 as the most highly upregulated gene in HCC among the 43 bromodomain-containing genes. Upregulation of BRPF1 was significantly associated with poorer patient survival. Therefore, targeting BRPF1 may be an approach for HCC treatment. Previously, several potential inhibitors of BRPF1 bromodomain have been discovered. However, due to the limited clinical success of the current inhibitors, we aim to search for new inhibitors with high affinity and specificity for the BRPF1 bromodomain. In this study, we utilized high-throughput virtual screening methods to screen synthetic and natural compound databases against the BRPF1 bromodomain. In addition, we used machine learning-based QSAR modeling to predict the IC50 values of the selected BRPF1 bromodomain inhibitors. Extensive MD simulations were used to calculate the binding free energies of BRPF1 bromodomain and inhibitor complexes. Using this approach, we identified four lead scaffolds with a similar or better binding affinity towards the BRPF1 bromodomain than the previously reported inhibitors. Overall, this study discovered some promising compounds that have the potential to act as potent BRPF1 bromodomain inhibitors.

Cysteine protease attribute of eukaryotic ribosomal protein S4.

BACKGROUND: Ribosomal proteins often carry out extraribosomal functions. The protein S4 from the smaller subunit of Escherichia coli, for instance, regulates self synthesis and acts as a transcription factor. In humans, S4 might be involved in Turner syndrome. Recent studies also associate many ribosomal proteins with malignancy, and cell death and survival. The list of extraribosomal functions of ribosomal proteins thus continues to grow. METHODS: Enzymatic action of recombinant wheat S4 on fluorogenic peptide substrates Ac-XEXD↓-AFC (N-acetyl-residue-Glu-residue-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR↓-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin) as well as proteins has been examined under a variety of solution conditions. RESULTS: Eukaryotic ribosomal protein S4 is an endoprotease exhibiting all characteristics of cysteine proteases. The K(m) value for the cleavage of Z-FR↓-AMC by a cysteine mutant (C41F) is about 70-fold higher relative to that for the wild-type protein under identical conditions, implying that S4 is indeed a cysteine protease. Interestingly, activity responses of the S4 protein and caspases toward environmental parameters, including pH, temperature, ionic strength, and Mg(2+) and Zn(2+) concentrations, are quite similar. Respective kinetic constants for their cleavage action on Ac-LEHD↓-AFC are also similar. However, S4 cannot be a caspase, because unlike the latter it also hydrolyzes the cathepsin substrate Z-FR↓-AMC. GENERAL SIGNIFICANCE: The eukaryotic S4 is a generic cysteine protease capable of hydrolyzing a broad spectrum of synthetic substrates and proteins. The enzyme attribute of eukaryotic ribosomal protein S4 is a new phenomenon. Its possible involvement in cell growth and proliferations are presented in the light of known extraribosomal roles of ribosomal proteins.

Identification of novel natural product inhibitors of BRD4 using high throughput virtual screening and MD simulation.

Bromodomains are evolutionarily conserved structural motifs that recognize acetylated lysine residues on histone tails. They play a crucial role in shaping chromatin architecture and regulating gene expression in various biological processes. Mutations in bromodomains containing proteins lead to multiple human diseases, which makes them attractive target for therapeutic intervention. Extensive studies have been done on BRD4 as a target for several cancers, such as Acute Myeloid Leukemia (AML) and Burkitt Lymphoma. Several potential inhibitors have been identified against the BRD4 bromodomain. However, most of these inhibitors have drawbacks such as non-specificity and toxicity, decreasing their appeal and necessitating the search for novel non-toxic inhibitors. This study aims to address this need by virtually screening natural compounds from the NPASS database against the Kac binding site of BRD4-BD1 using high throughput molecular docking followed by similarity clustering, pharmacokinetic screening, MD simulation and MM-PBSA binding free energy calculations. Using this approach, we identified five natural product inhibitors having a similar or better binding affinity to the BRD4 bromodomain compared to JQ1 (previously reported inhibitor of BRD4). Further systematic analysis of these inhibitors resulted in the top three hits: NPC268484 (Palodesangren-B), NPC295021 (Candidine) and NPC313112 (Buxifoliadine-D). Collectively, our in silico results identified some promising natural products that have the potential to act as potent BRD4-BD1 inhibitors and can be considered for further validation through future in vitro and in vivo studies.Communicated by Ramaswamy H. Sarma.

Insulin fibril inhibition using glycopolymeric nanoassemblies.

To address the obstacles in insulin protein homeostasis leading to the formation of neurotoxic amyloid plaques associated with different diseases, herein we have synthesized block copolymers using the reversible addition-fragmentation chain transfer (RAFT) polymerization method, composed of tert-butoxycarbonyl (Boc) protected leucine and acetyl (Ac) protected glucose pendant moieties, respectively. Selective or dual deprotection of Boc- and Ac-groups from leucine and/or glucose moieties resulted in amphiphilic polymers, which self-assembled into nanoaggregates in aqueous medium, confirmed by critical aggregation concentration (CAC) determination, dynamic light scattering (DLS) and transmission electron microscopy (TEM). These glycopolymeric nanoassemblies were used to study the inhibition rates of insulin fibrillation and were found to impede the fibrillation of the insulin protein. Using several biophysical techniques, we observed that hydrophobic, electrostatic, and hydrogen bonding interactions were responsible for binding the insulin monomer/oligomer with various glycopolymeric aggregates, inhibiting insulin fibrillation. Tyrosine (Tyr) and Nile red (NR) fluorescence measurements manifested the hydrophobic interactions, whereas temperature-dependent fluorescence and isothermal titration calorimetry (ITC) measurements revealed respectively the hydrogen bonding and electrostatic interactions involved in the inhibition process of insulin amyloid formation. Molecular dynamics simulations further confirmed the involvement of different interactions among polymer-protein residues in averting the fibrillation process.

RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug.

BACKGROUND: Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. RESULTS: We performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. CONCLUSIONS: We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C. lectularius. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets such as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in C. lectularius.

Cloning, Escherichia coli expression, purification, characterization, and enzyme assay of the ribosomal protein S4 from wheat seedlings (Triticum vulgare).

S4 is a paradigm of ribosomal proteins involved in multifarious activities both within and outside the ribosome. For a detailed biochemical and structural investigations of eukaryotic S4, the wheat S4 gene has been cloned and expressed in Escherichia coli, and the protein purified to a high degree of homogeneity. The 285-residue recombinant protein containing an N-terminal His(6) tag along with fourteen additional residues derived from the cloning vector is characterized by a molecular mass of 31981.24 Da. The actual sequence of 265 amino acids having a molecular mass of 29931 Da completely defines the primary structure of wheat S4. Homology modeling shows a bi-lobed protein topology arising from folding of the polypeptide into two domains, consistent with the fold topology of prokaryotic S4. The purified protein is stable and folded since it can be reversibly unfolded in guanidinium hydrochloride, and is capable of hydrolyzing cysteine protease-specific peptide-based fluorescence substrates, including Ac-DEVD-AFC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin).