Methoprene application and diet protein supplementation to male melon fly,Bactrocera cucurbitae,modifies female remating behavior

Methoprene (an analogue of juvenile hormone) application and feeding on a protein diet is known to enhance male melon fly, Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), mating success. In this study, we investigated the effect of these treatments on male B. cucurbitae's ability to inhibit female remating. While 14‐d‐old females were fed on protein diet, 6‐d‐old males were exposed to one of the following treatments: (i) topical application of methoprene and fed on a protein diet; (ii) no methoprene but fed on a protein diet; (iii) methoprene and sugar‐fed only; and (iv) sugar‐fed, 14‐d‐old males acted as controls. Treatments had no effect on a male's ability to depress the female remating receptivity in comparison to the control. Females mated with protein‐deprived males showed higher remating receptivity than females first mated with protein‐fed males. Methoprene and protein diet interaction had a positive effect on male mating success during the first and second mating of females. Significantly more females first mated with sugar‐fed males remated with protein‐fed males and females first mated with methoprene treated and protein‐fed males were more likely to remate with similarly treated males. Females mating latency (time to start mating) was significantly shorter with protein‐fed males, and mating duration was significantly longer with protein‐fed males compared with protein‐deprived males. These results are discussed in the context of methoprene and/or dietary protein as prerelease treatment of sterile males in area‐wide control of melon fly integrating the sterile insect technique (SIT).     

The possible roles of B‐cell novel protein‐1 (BCNP1) in cellular signalling pathways and in cancer

B‐cell novel protein‐1 (BCNP1) or Family member of 129C (FAM129C) was identified as a B‐cell‐specific plasma‐membrane protein. Bioinformatics analysis predicted that BCNP1 might be heavily phosphorylated. The BCNP1 protein contains a pleckstrin homology (PH) domain, two proline‐rich (PR) regions and a Leucine Zipper (LZ) domain suggesting that it may be involved in protein‐protein interactions. Using The Cancer Genome Atlas (TCGA) data sets, we investigated the correlation of alteration of the BCNP1 copy‐number changes and mutations in several cancer types. We also investigated the function of BCNP1 in cellular signalling pathways. We found that BCNP1 is highly altered in some types of cancers and that BCNP1 copy‐number changes and mutations co‐occur with other molecular alteration events for TP53 (tumour protein P53), PIK3CA (Phosphatidylinositol‐4,5‐Bisphosphate 3‐Kinase, Catalytic Subunit Alpha), MAPK1 (mitogen‐activated protein kinase‐1; ERK: extracellular signal regulated kinase), KRAS (Kirsten rat sarcoma viral oncogene homolog) and AKT2 (V‐Akt Murine Thymoma Viral Oncogene Homolog 2). We also found that PI3K (Phoshoinositide 3‐kinase) inhibition and p38 MAPK (p38 mitogen‐activated protein kinase) activation leads to reduction in phosphorylation of BCNP1 at serine residues, suggesting that BCNP1 phosphorylation is PI3K and p38MAPK dependent and that it might be involved in cancer. Its degradation depends on a proteasome‐mediated pathway.     

Deficiency of terminal ADP‐ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

Adenosine diphosphate (ADP)‐ribosylation is a post‐translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP‐ribosylation reactions are the poly(ADP‐ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP‐ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP‐ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP‐ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP‐interacting protein that removes mono(ADP‐ribosyl)ation on glutamate amino acid residues in PARP‐modified proteins. X‐ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl‐(ADP‐ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP‐ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.     

Fusion order controls expression level and activity of elastin‐like polypeptide fusion proteins

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin‐like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C‐terminus of the target protein (protein‐ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP‐protein constructs have a lower specific activity than protein‐ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein‐ELP and ELP‐protein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N‐terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C‐terminal end of the protein.     

A redox‐linked novel pathway for arsenic‐mediated RET tyrosine kinase activation

We examined the biochemical effects of arsenic on the activities of RET proto‐oncogene (c‐RET protein tyrosine kinases) and RET oncogene (RET‐MEN2A and RET‐PTC1 protein tyrosine kinases) products. Arsenic activated c‐RET kinase with promotion of disulfide bond‐mediated dimerization of c‐RET protein. Arsenic further activated RET‐MEN2A kinase, which was already 3‐ to 10‐fold augmented by genetic mutation compared with c‐RET kinase activity, with promotion of disulfide bond‐mediated dimerization of RET‐MEN2A protein (superactivation). Arsenic also increased extracellular domain‐deleted RET‐PTC1 kinase activity with promotion of disulfide bond‐mediated dimerization of RET‐PTC1 protein. Arsenic increased RET‐PTC1 kinase activity with cysteine 365 (C365) replaced by alanine with promotion of dimer formation but not with cysteine 376 (C376) replaced by alanine. Our results suggest that arsenic‐mediated regulation of RET kinase activity is dependent on conformational change of RET protein through modulation of a special cysteine sited at the intracellular domain in RET protein (relevant cysteine of C376 in RET‐PTC1 protein). Moreover, arsenic enhanced the activity of immunoprecipitated RET protein with increase in thiol‐dependent dimer formation. As arsenic (14.2 µM) was detected in the cells cultured with arsenic (100 µM), direct association between arsenic and RET in the cells might modulate dimer formation. Thus, we demonstrated a novel redox‐linked mechanism of activation of arsenic‐mediated RET proto‐oncogene and oncogene products. J. Cell. Biochem. 110: 399–407, 2010. © 2010 Wiley‐Liss, Inc.     

Matrix‐glycoprotein interactions required for budding of a plant nucleorhabdovirus and induction of inner nuclear membrane invagination

Nucleorhabdoviruses such as Sonchus yellow net virus (SYNV) replicate in the nuclei and undergo morphogenesis at the inner nuclear membrane (IM) in plant cells. Mature particles are presumed to form by budding of the Matrix (M) protein‐nucleocapsid complexes through host IMs to acquire host phospholipids and the surface glycoproteins (G). To address mechanisms underlying nucleorhabdovirus budding, we generated recombinant SYNV G mutants containing a truncated amino‐terminal (NT) or carboxyl‐terminal (CT) domain. Electron microscopy and sucrose gradient centrifugation analyses showed that the CT domain is essential for virion morphogenesis whereas the NT domain is also required for efficient budding. SYNV infection induces IM invaginations that are thought to provide membrane sites for virus budding. We found that in the context of viral infections, interactions of the M protein with the CT domain of the membrane‐anchored G protein mediate M protein translocation and IM invagination. Interestingly, tethering the M protein to endomembranes, either by co‐expression with a transmembrane G protein CT domain or by artificial fusion with the G protein membrane targeting sequence, induces IM invagination in uninfected cells. Further evidence to support functions of G‐M interactions in virus budding came from dominant negative effects on SYNV‐induced IM invagination and viral infections that were elicited by expression of a soluble version of the G protein CT domain. Based on these data, we propose that cooperative G‐M interactions promote efficient SYNV budding.     

Are endoplasmic reticulum subdomains shaped by asymmetric distribution of phospholipids? Evidence from a C. elegans model system

Physical contact between organelles are widespread, in part to facilitate the shuttling of protein and lipid cargoes for cellular homeostasis. How do protein‐protein and protein‐lipid interactions shape organelle subdomains that constitute contact sites? The endoplasmic reticulum (ER) forms extensive contacts with multiple organelles, including lipid droplets (LDs) that are central to cellular fat storage and mobilization. Here, we focus on ER‐LD contacts that are highlighted by the conserved protein seipin, which promotes LD biogenesis and expansion. Seipin is enriched in ER tubules that form cage‐like structures around a subset of LDs. Such enrichment is strongly dependent on polyunsaturated and cyclopropane fatty acids. Based on these findings, we speculate on molecular events that lead to the formation of seipin‐positive peri‐LD cages in which protein movement is restricted. We hypothesize that asymmetric distribution of specific phospholipids distinguishes cage membrane tubules from the bulk ER.     

l-Arginine hydrochloride increases the solubility of folded and unfolded recombinant plasminogen activator rPA

L ‐Arginine hydrochloride (L ‐ArgHCl) was found to be an effective enhancer for in vitro protein refolding more than two decades ago. A detailed understanding of the mechanism of action, by which L ‐ArgHCl as co‐solvent is capable to effectively suppress protein aggregation, while protein stability is preserved, has remained elusive. Concepts for the effects of co‐solvents, which have been established over the last decades, were found to be insufficient to completely explain the effects of L ‐ArgHCl on protein refolding. In this article, we present data, which clearly establish that L ‐ArgHCl acts on the equilibrium solubility of the native model protein recombinant plasminogen activator (rPA), while for S‐carboxymethylated rPA (IAA‐rPA) that served as a model protein for denatured protein states, equilibrium solubilities could not be obtained. Solid to solute free transfer energies for native rPA were lowered by up to 14 kJ mol^‐1 under the tested conditions. This finding is in marked contrast to a previously proposed model in which L ‐ArgHCl acts as a neutral crowder which exclusively has an influence on the stability of the transition state of aggregation. The effects on the apparent solubility of IAA‐rPA, as well as on the aggregation kinetics of all studied protein species, that were observed in the present work could tentatively be explained within the framework of a nucleation‐aggregation scheme, in which L ‐ArgHCl exerts a strong effect on the pre‐equilibria leading to formation of the aggregation seed.     

The Nucleotide‐Dependent Interactome of Rice Heterotrimeric G‐Protein α ‐Subunit

The rice heterotrimeric G‐protein complex, a guanine‐nucleotide‐dependent on‐off switch, mediates vital cellular processes and responses to biotic and abiotic stress. Exchange of bound GDP (resting state) for GTP (active state) is spontaneous in plants including rice and thus there is no need for promoting guanine nucleotide exchange in vivo as a mechanism for regulating the active state of signaling as it is well known for animal G signaling. As such, a master regulator controlling the G‐protein activation state is unknown in plants. Therefore, an ab initio approach is taken to discover candidate regulators. The rice Gα subunit (RGA1) is used as bait to screen for nucleotide‐dependent protein partners. A total of 264 proteins are identified by tandem mass spectrometry of which 32 were specific to the GDP‐bound inactive state and 22 specific to the transition state. Approximately, 10% are validated as previously identified G‐protein interactors.     

Distinct roles of ATR and DNA‐PKcs in triggering DNA damage responses in ATM‐deficient cells

The cellular response to DNA double‐strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell‐cycle‐dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell‐cycle‐independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM‐deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM‐deficient cells enforce an early G2/M checkpoint that is ATR‐dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM‐deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP‐1), a protein involved in chromatin remodelling, is mediated by DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in a spatio‐temporal manner in addition to ATM. We posit that ATM substrates involved in cell‐cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA‐PKcs in addition to ATM.     

Purification and Partial Characterization of a Novel Neurotoxic Protein from Eggs of Black Widow Spiders (Latrodectus tredecimguttatus)

Up to now, there have been a few reports on the toxic components purified from black widow spider (Latrodectus tredecimguttatus) eggs. In the present study, a novel neurotoxic protein was purified from the eggs by gel filtration combined with ion‐exchange chromatography. Its molecular weight was 23.752 kDa determined by electrospray mass spectrometry. The protein could block the neuromuscular transmission in mouse‐isolated phrenic nerve‐hemidiaphragm preparations completely in a reversible manner and activate tetrodotoxin‐sensitive sodium current in rat dorsal root ganglion cells. The N‐terminal sequence of the protein was identified by the Edman degradation to be N‐S‐I‐A‐D‐D‐R‐Y‐R‐W‐P‐G‐Y‐P‐G‐A‐G‐L‐I‐P‐Y‐I‐I‐D‐S—. When the sequence was used to search against protein database with a sequence query in Mascot engine there was no matched sequence or protein whereas the Basic Local Alignment Search Tool (BLAST) analysis indicated that no significant similarity was found. These results demonstrated that the protein (named Latroeggtoxin‐I) is a novel neurotoxic protein purified from the eggs of black widow spiders. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:337‐342, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21493     

Comparing the binding properties of peptides mimicking the Envelope protein of SARS‐CoV and SARS‐CoV‐2 to the PDZ domain of the tight junction‐associated PALS1 protein

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS‐CoV and SARS‐CoV‐2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ‐binding motif at its C‐terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS‐CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS‐CoV and SARS‐CoV‐2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS‐CoV‐2 compared to SARS‐CoV may rely on the increased affinity of its Envelope protein for PALS1.     

Sequence and structural analysis of fibronectin‐binding protein reveals importance of multiple intrinsic disordered tandem repeats

The location of certain amino acid sequences like repeats along the polypeptide chain is very important in the context of forming the overall shape of the protein molecule which in fact determines its function. In gram‐positive bacteria, fibronectin‐binding protein (FnBP) is one such repeat containing protein, and it is a cell wall‐attached protein responsible for various acute infections in human. Several studies on sequence, structure, and function of fibronectin‐binding regions of FnBPs were reported; however, no detailed study was carried out on the full‐length protein sequence. In the present study, we have made a thorough sequence and structure analysis on FnBP_A of Staphylococcus aureus and explored the presence of dual ligand‐binding ability of fibrinogen (fg)‐binding region and its molecular recognition processes. Multiple sequence alignment and protein‐protein docking analysis reveal the regions which are likely involved in dual ligand binding. Further analysis of docking of FnBP_A fg‐binding region and fn N‐terminal modules suggests that if the latter binds to the fg‐binding region of FnBP_A, it would inhibit the subsequent binding of fg because of steric hindrance. The sequence analysis further suggests that the abundance of disorder promoting residue glutamic acid and dual personality (both order/disorder promoting) residue threonine in tandem repeats of FnBP_A and B proteins possibly would help the molecule to undergo a conformational change while binding with fn by β‐zipper mechanism. The segment‐based power spectral analysis was carried out which helps to understand the distribution of hydrophobic residues along the sequence particularly in intrinsic disordered tandem repeats. The results presented here will help to understand the role of internal repeats and intrinsic disorder in the molecular recognition process of a pathogenic cell surface protein.     

Tissue microarray profiling in human heart failure

Tissue MicroArrays (TMAs) are a versatile tool for high‐throughput protein screening, allowing qualitative analysis of a large number of samples on a single slide. We have developed a customizable TMA system that uniquely utilizes cryopreserved human cardiac samples from both heart failure and donor patients to produce formalin‐fixed paraffin‐embedded sections. Confirmatory upstream or downstream molecular studies can then be performed on the same (biobanked) cryopreserved tissue. In a pilot study, we applied our TMAs to screen for the expression of four‐and‐a‐half LIM‐domain 2 (FHL2), a member of the four‐and‐a‐half LIM family. This protein has been implicated in the pathogenesis of heart failure in a variety of animal models. While FHL2 is abundant in the heart, not much is known about its expression in human heart failure. For this purpose, we generated an affinity‐purified rabbit polyclonal anti‐human FHL2 antibody. Our TMAs allowed high‐throughput profiling of FHL2 protein using qualitative and semiquantitative immunohistochemistry that proved complementary to Western blot analysis. We demonstrated a significant relative reduction in FHL2 protein expression across different forms of human heart failure.     

Refining near‐native protein–protein docking decoys by local resampling and energy minimization

How to refine a near‐native structure to make it closer to its native conformation is an unsolved problem in protein‐structure and protein–protein complex‐structure prediction. In this article, we first test several scoring functions for selecting locally resampled near‐native protein–protein docking conformations and then propose a computationally efficient protocol for structure refinement via local resampling and energy minimization. The proposed method employs a statistical energy function based on a Distance‐scaled Ideal‐gas REference state (DFIRE) as an initial filter and an empirical energy function EMPIRE (EMpirical Protein‐InteRaction Energy) for optimization and re‐ranking. Significant improvement of final top‐1 ranked structures over initial near‐native structures is observed in the ZDOCK 2.3 decoy set for Benchmark 1.0 (74% whose global rmsd reduced by 0.5 Å or more and only 7% increased by 0.5 Å or more). Less significant improvement is observed for Benchmark 2.0 (38% versus 33%). Possible reasons are discussed. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Analysis of differential proteins in two different wing‐types of female Nilaparvata lugens

Nilaparvata lugens (stal) is a rice pest and contains long‐winged and short‐winged varieties, called the wing differentiation. This study compared the protein profiles of the two wing‐types in females and two wing‐disc types 5th‐instar females by two‐dimensional electrophoresis analysis. We detected 172 and 174 protein spots in adults and 5th‐instar nymphs, respectively. The number of proteins with higher content in the long‐winged (disc) individuals is much more than that in the short‐winged (disc) individuals. A total of 32 differential protein spots were found, of which 20 were successfully identified. Their main function is about catabolic process, fiber and nucleoside binding, and they constitute 52 protein–protein interactions, which is around the glycolysis as the core. These results enrich the research on the protein Level in wing development, and provide more references for future studies.     

Isatin‐induced increase in the affinity of human ferrochelatase and adrenodoxin reductase interaction

Isatin (indol‐2,3‐dione) is an endogenous non‐peptide regulator exhibiting a wide range of biological and pharmacological activities, which are poorly characterized in terms of their molecular mechanisms. Identification of many isatin‐binding proteins in the mammalian brain and liver suggests that isatin may influence their functions. We have hypothesized that besides direct action on particular protein targets, isatin can act as a regulator of protein–protein interactions (PPIs). In this surface plasmon resonance‐based biosensor study we have found that physiologically relevant concentrations of isatin (25‐100 μM) increase affinity of interactions between human recombinant ferrochelatase (FECH) and NADPH‐dependent adrenodoxin reductase (ADR). In the presence of increasing concentrations of isatin the Kd values demonstrated a significant (up to 6‐fold) decrease. It is especially important that the interaction of isatin with each individual protein (FECH, ADR) was basically negligible and therefore could not contribute to the observed effect. This effect was specific only for the FECH/ADR complex formation and was not observed for other protein complexes studied: FECH/cytochrome b5(CYB5A) and FECH/SMAD4.