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Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Bromodomains are evolutionarily conserved reader modules that recognize acetylated lysine residues on the histone tails to facilitate gene transcription. The bromodomain and PHD finger containing protein 3 (BRPF3) is a scaffolding protein that forms a tetrameric complex with HBO1 histone acetyltransferase (HAT) and two other subunits, which is known to regulate the HAT activity and substrate specificity. However, its molecular mechanism, histone ligands, and biological functions remain unknown. Herein, we identify mono‐ (H4K5ac) and di‐ (H4K5acK12ac) acetylated histone peptides as novel interacting partners of the BRPF3 bromodomain. Consistent with this, pull‐down assays on purified histones from human cells confirm the interaction of BRPF3 bromodomain with acetylated histone H4. Further, MD simulation studies highlight the binding mode of acetyllysine (Kac) and the stability of bromodomain‐histone peptide complexes. Collectively, our findings provide a key insight into how histone targets of the BRPF3 bromodomain direct the recruitment of HBO1 complex to chromatin for downstream transcriptional regulation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACT Ceruloplasmin, a multicopper oxidase essential for copper metabolism and oxidative defense, from blood serum of domestic goats (Capra aegagrus hircus) has been purified to homogeneity by using three steps of column chromatography. The purified goat ceruloplasmin (gCP) protein has an estimated molecular weight of approximately 141 kDa as determined by SDS‐PAGE. ICP‐AES analysis indicates that each molecule of the protein contains 5.6 ± 0.2 atoms of copper. The optimum pH of catalysis for gCP was found to be pH 5.5. Using the Hill equation, the Km and Vmax values for gCP are estimated to be 0.41 ± 0.01 mM and 0.35 ± 0.01 μmol/min/mg, respectively. The enzyme has an average of 3.3 ± 0.33 substrate binding sites per molecule. Both, circular dichroism (CD) spectroscopy studies and computational analysis using PDBsum reveals that gCP is primarily composed of β‐strands distinguishing it from the α‐helix‐rich human ceruloplasmin. Also, computational analysis of gCP reveals six copper‐binding sites, composed of histidine (his), methionine (Met), and cysteine (cys). Cyclic voltammetry studies of gCP immobilized on a glassy carbon electrode indicate the presence of Type 1 and Type 2/3 copper centers within the protein identified by two unique redox potential values of +294 mV and −250 mV versus the normal hydrogen electrode (NHE), respectively. The positive redox potential site that probably acts as the electron‐transfer center had a slightly lower redox potential compared to that reported in other ceruloplasmin. The negative redox potential site of the protein possibly corresponds to the catalytic site containing trinuclear copper center proposed earlier. The redox potential of the Type 1 center aligns closely with azurin, suggesting evolutionary homology and suitability for direct electron transfer (DET). The distinct electrochemical properties of gCP may enhance applications in biofuel cells, biosensors, and other bioelectronic devices due to its efficient electron‐transfer capabilities. This study provides valuable insights into the functional versatility of ceruloplasmin across species and highlights the biotechnological potential of this unique variant. [ABSTRACT FROM AUTHOR]