Mechanism of activation and inhibition of the HER4/ErbB4 kinase

HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.     

A conserved anti-repressor controls horizontal gene transfer by proteolysis

The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in the Bacillus subtilis chromosome. The SOS response and the RapI-PhrI sensory system activate ICEBs1 gene expression, excision and transfer by inactivating the ICEBs1 repressor protein ImmR. Although ImmR is similar to many characterized phage repressors, we found that, unlike these repressors, inactivation of ImmR requires an ICEBs1-encoded anti-repressor ImmA (YdcM). ImmA was needed for the degradation of ImmR in B. subtilis. Coexpression of ImmA and ImmR in Escherichia coli or co-incubation of purified ImmA and ImmR resulted in site-specific cleavage of ImmR. Homologues of immR and immA are found in many mobile genetic elements. We found that the ImmA homologue encoded by B. subtilis phage phi105 is required for inactivation of the phi105 repressor (an ImmR homologue). ImmA-dependent proteolysis of ImmR repressors may be a conserved mechanism for regulating horizontal gene transfer.     

Protein folding following synthesis in vitro and in vivo: association of newly synthesized protein with 50S subunit of E. coli ribosome

In the accompanying paper, it was shown that a protein, while reverting to native form from the unfolded state in vitro with the help of bacterial 70S ribosome, split the latter into its subunits (50S and 30S) and remains associated with the 50S subunit. Here, we follow the fate of nascent proteins both in case of in vivo and in vitro translation system. The newly synthesised protein was found to associate with the 50S subunit in both the cases.     

Estimating odds ratios in genome scans: an approximate conditional likelihood approach

In modern whole-genome scans, the use of stringent thresholds to control the genome-wide testing error distorts the estimation process, producing estimated effect sizes that may be on average far greater in magnitude than the true effect sizes. We introduce a method, based on the estimate of genetic effect and its standard error as reported by standard statistical software, to correct for this bias in case-control association studies. Our approach is widely applicable, is far easier to implement than competing approaches, and may often be applied to published studies without access to the original data. We evaluate the performance of our approach via extensive simulations for a range of genetic models, minor allele frequencies, and genetic effect sizes. Compared to the naive estimation procedure, our approach reduces the bias and the mean squared error, especially for modest effect sizes. We also develop a principled method to construct confidence intervals for the genetic effect that acknowledges the conditioning on statistical significance. Our approach is described in the specific context of odds ratios and logistic modeling but is more widely applicable. Application to recently published data sets demonstrates the relevance of our approach to modern genome scans.     

StAR-like activity and molten globule behavior of StARD6, a male germ-line protein

The steroidogenic acute regulatory protein (StAR) belongs to a family of 15 StAR-related lipid transfer (START) domain proteins termed StARD1-StARD15. StAR (StARD1) induces adrenal and gonadal steroidogenesis by moving cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane by an unclear process that involves conformational changes that have been characterized as a molten globule transition. We expressed, purified, and assessed the activity and cholesterol-binding behavior of StARD1 and StARD3-D7, showing that StARD6 had activity equal to StARD1, whereas StARD4, D5, and D7 had little or no activity with adrenal mitochondria in vitro. Partial proteolysis examined by mass spectrometry suggests that StARD6 has a protease-sensitive C-terminus, similar to but smaller than that of StARD1. Experiments using urea denaturation, stopped-flow kinetics and measurements of mitochondrial membrane association suggests that StARD1 and StARD6 both unfold and refold slowly with similar kinetic patterns. Isothermal titration calorimetry suggests that StARD6 interacts with mitochondrial membranes as well as or better than StARD1. Computational modeling of StARD6 suggests that it has a similar fold to StARD1, with a hydrophobic sterol-binding pocket and a unique C-terminal extension. StARD6, which is expressed only in male germ-line cells, thus exhibits biological and biophysical properties that imply a role in steroidogenesis.     

Study of laccase production by Pleurotus ostreatus in a 5 l bioreactor and application of the enzyme to determine the antioxidant concentration of human plasma

Aims: To achieve high laccase production from Pleurotus ostreatus in a bench top bioreactor and to utilize the enzyme for determination of the total antioxidant concentration (TAC) of human plasma. Methods and Results: Laccase production by P. ostreatus studied in a benchtop bioreactor was as high as, 874·0 U ml^?1 in presence of copper sulfate. The enzyme was used to replace metmyoglobin and hydrogen peroxide for the estimation of TAC in human plasma. The trolox equivalent antioxidant concentrations determined by the laccase‐based method and metmyoglobin method ranged from 1·63 ± 0·011 to 1·80 ± 0·006 mmol l^?1 and from 1·41 ± 0·004 to 1·51 ± 0·008 mmol l^?1 plasma, respectively. Conclusions: Pleurotus ostreatus produced high amount of extracellular laccase in a benchtop bioreactor. The enzyme can be used to assay TAC of blood plasma without the interference encountered with the hydrogen peroxide and metmyoglobin mediated assay method. Significance and Impact of the Study: Laccase production by P. ostreatus obtained in this study was the highest among all reported laccase producing white‐rot fungi. Moreover, an accurate laccase‐based assay method was developed for detection of TAC in human plasma.     

Differential modification of p27Kip1 controls its cyclin D-cdk4 inhibitory activity

Whether p27 is a cyclin D-cdk4/6 inhibitor or not is controversial, and how it might switch between these two modes is unknown. Arguing for a two-state mechanism, we show that p27 bound to cyclin D-cdk4 can be both inhibitory and noninhibitory, due to its differential-growth-state-dependent tyrosine phosphorylation. We found that p27 from proliferating cells was noninhibitory but that p27 from arrested cells was inhibitory, and the transition from a bound noninhibitor to a bound inhibitor was not due to an increase in p27 concentration. Rather, two tyrosine residues (Y88 and Y89) in p27's cdk interaction domain were phosphorylated preferentially in proliferating cells, which converted p27 to a noninhibitor. Concordantly, mutation of these sites rendered p27 resistant to phosphorylation and locked it into the bound-inhibitor mode in vivo and in vitro. Y88 was directly phosphorylated in vitro by the tyrosine kinase Abl, which converted p27 to a cdk4-bound noninhibitor. These data show that the growth-state-dependent tyrosine phosphorylation of p27 modulates its inhibitory activity in vivo.     

Analyzing text recognition from tactually evoked EEG

Tactual exploration of objects produce specific patterns in the human brain and hence objects can be recognized by analyzing brain signals during tactile exploration. The present work aims at analyzing EEG signals online for recognition of embossed texts by tactual exploration. EEG signals are acquired from the parietal region over the somatosensory cortex of blindfolded healthy subjects while they tactually explored embossed texts, including symbols, numbers, and alphabets. Classifiers based on the principle of supervised learning are trained on the extracted EEG feature space, comprising three features, namely, adaptive autoregressive parameters, Hurst exponents, and power spectral density, to recognize the respective texts. The pre-trained classifiers are used to classify the EEG data to identify the texts online and the recognized text is displayed on the computer screen for communication. Online classifications of two, four, and six classes of embossed texts are achieved with overall average recognition rates of 76.62, 72.31, and 67.62% respectively and the computational time is less than 2 s in each case. The maximum information transfer rate and utility of the system performance over all experiments are 0.7187 and 2.0529 bits/s respectively. This work presents a study that shows the possibility to classify 3D letters using tactually evoked EEG. In future, it will help the BCI community to design stimuli for better tactile augmentation n also opens new directions of research to facilitate 3D letters for visually impaired persons. Further, 3D maps can be generated for aiding tactual BCI in teleoperation.     

Induced resistance in cells exposed to repeated low doses of H2O2 involves enhanced activity of antioxidant enzymes

We have derived cells from the Chinese hamster V79 cell line by conditioning them with repeated low doses of hydrogen peroxide (H(2)O(2)). This mimics the physiological condition where cells are repeatedly exposed to low levels of oxidants. In an attempt to characterize such cells, we have exposed both conditioned cells (V79(C)) and the parental V79 cells (V79(P)) to different types of cytotoxic agents and compared their sensitivity to cell killing. The V79(C) cells were found to be stably resistant to killing by agents that produced toxicity through oxidative stress, e.g. H(2)O(2) and cisplatin. It was also found that the lipid peroxidation produced by these agents were considerably lower in the V79(C) cells. Thus, the difference in sensitivity could be due to lesser extent of damage to these cells. V79(C) cells had greater antioxidant defense through higher GSH content and greater activity of enzymes such as Cu-Zn superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which provided protection from damage. Enzyme activities were also assayed at different times after treatment with various cytotoxic agents; there was a relatively large increase in SOD activity which perhaps plays a key role in determining the resistance of the V79(C) cells to killing.