Quantitative proteomics identifies the Myb-binding protein p160 as a novel target of the von Hippel-Lindau tumor suppressor

Background

The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex, which is best understood as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the α subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. However, several lines of evidence suggest that there are unidentified substrates or targets for VHL that play important roles in tumor suppression.

Methodology/Principal Findings

Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL.

Conclusions/Significance

A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates. The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases.     

Identification of lipocalin-2 as a PKCδ phosphorylation substrate in neutrophils

Background

PKCδ expressed in neutrophils is implicated in promoting reperfusion injury after ischemic stroke. To understand the molecular and cellular actions of PKCδ, we employed a chemical-genetics approach to identify PKCδ substrates in neutrophils.

Results

We recently generated knock-in mice endogenously expressing analog-specific PKCδ (AS-PKCδ) that can utilize ATP analogs as phosphate donors. Using neutrophils isolated from the knock-in mice, we identified several PKCδ substrates, one of which was lipocalin-2 (LCN2), which is an iron-binding protein that can trigger apoptosis by reducing intracellular iron concentrations. We found that PKCδ phosphorylated LCN2 at T115 and this phosphorylation was reduced in Prkcd^−/− mice. PKCδ colocalized with LCN2 in resting and stimulated neutrophils. LCN2 release from neutrophils after cerebral ischemia was reduced in PKCδ null mice.

Conclusions

These findings suggest that PKCδ phosphorylates LCN2 and mediates its release from neutrophils during ischemia-reperfusion injury.     

GSK3 regulates mitotic chromosomal alignment through CRMP4

Background

Glycogen Synthase Kinase 3 (GSK3) has been implicated in regulating chromosomal alignment and mitotic progression but the physiological substrates mediating these GSK3-dependent effects have not been identified. Collapsin Response Mediator Protein 4 (CRMP4) is a cytosolic phosphoprotein known to regulate cytoskeletal dynamics and is a known physiological substrate of GSK3. In this study, we investigate the role of CRMP4 during mitosis.

Methodology and Principal Findings

Here we demonstrate that during mitosis CRMP4 phosphorylation is regulated in a GSK3-dependent manner. We show that CRMP4 localizes to spindle microtubules during mitosis and loss of CRMP4 disrupts chromosomal alignment and mitotic progression. The effect of CRMP4 on chromosomal alignment is dependent on phosphorylation by GSK3 identifying CRMP4 as a critical GSK3 substrate during mitotic progression. We also provide mechanistic data demonstrating that CRMP4 regulates spindle microtubules consistent with its known role in the regulation of the microtubule cytoskeleton.

Conclusion and Significance

Our findings identify CRMP4 as a key physiological substrate of GSK3 in regulating chromosomal alignment and mitotic progression through its effect on spindle microtubules.     

Correlated Mutation Analysis on the Catalytic Domains of Serine/Threonine Protein Kinases

Background

Protein kinases (PKs) have emerged as the largest family of signaling proteins in eukaryotic cells and are involved in every aspect of cellular regulation. Great progresses have been made in understanding the mechanisms of PKs phosphorylating their substrates, but the detailed mechanisms, by which PKs ensure their substrate specificity with their structurally conserved catalytic domains, still have not been adequately understood. Correlated mutation analysis based on large sets of diverse sequence data may provide new insights into this question.

Methodology/Principal Findings

Statistical coupling, residue correlation and mutual information analyses along with clustering were applied to analyze the structure-based multiple sequence alignment of the catalytic domains of the Ser/Thr PK family. Two clusters of highly coupled sites were identified. Mapping these positions onto the 3D structure of PK catalytic domain showed that these two groups of positions form two physically close networks. We named these two networks as θ-shaped and γ-shaped networks, respectively.

Conclusions/Significance

The θ-shaped network links the active site cleft and the substrate binding regions, and might participate in PKs recognizing and interacting with their substrates. The γ-shaped network is mainly situated in one side of substrate binding regions, linking the activation loop and the substrate binding regions. It might play a role in supporting the activation loop and substrate binding regions before catalysis, and participate in product releasing after phosphoryl transfer. Our results exhibit significant correlations with experimental observations, and can be used as a guide to further experimental and theoretical studies on the mechanisms of PKs interacting with their substrates.     

Imaging β-Galactosidase Activity in Human Tumor Xenografts and Transgenic Mice Using a Chemiluminescent Substrate

Background

Detection of enzyme activity or transgene expression offers potential insight into developmental biology, disease progression, and potentially personalized medicine. Historically, the lacZ gene encoding the enzyme β-galactosidase has been the most common reporter gene and many chromogenic and fluorogenic substrates are well established, but limited to histology or in vitro assays. We now present a novel approach for in vivo detection of β-galactosidase using optical imaging to detect light emission following administration of the chemiluminescent 1,2-dioxetane substrate Galacto-Light PlusTM.

Methodology and Principal Findings

B-gal activity was visualized in stably transfected human MCF7-lacZ tumors growing in mice. LacZ tumors were identified versus contralateral wild type tumors as controls, based on two- to tenfold greater light emission following direct intra tumoral or intravenous administration of reporter substrate. The 1,2-dioxetane substrate is commercially available as a kit for microplate-based assays for β-gal detection, and we have adapted it for in vivo application. Typically, 100 µl substrate mixture was administered intravenously and light emission was detected from the lacZ tumor immediately with gradual decrease over the next 20 mins. Imaging was also undertaken in transgenic ROSA26 mice following subcutaneous or intravenous injection of substrate mixture.

Conclusion and Significance

Light emission was detectable using standard instrumentation designed for more traditional bioluminescent imaging. Use of 1,2-dioxetane substrates to detect enzyme activity offers a new paradigm for non-invasive biochemistry in vivo.     

The Association of BAG6 with SGTA and Tail-Anchored Proteins

Background

The BAG6 protein is a subunit of a heterotrimeric complex that binds a range of membrane and secretory protein precursors localized to the cytosol, enforcing quality control and influencing their subsequent fate.

Methodology and Principal Findings

BAG6 has an N-terminal ubiquitin-like domain, and a C-terminal Bcl-2-associated athanogene domain, separated by a large central proline-rich region. We have used in vitro binding approaches to identify regions of BAG6 important for its interactions with: i) the small-glutamine rich tetratricopeptide repeat-containing protein alpha (SGTA) and ii) two model tail-anchored membrane proteins as a paradigm for its hydrophobic substrates. We show that the BAG6-UBL is essential for binding to SGTA, and find that the UBL of a second subunit of the BAG6-complex, ubiquitin-like protein 4A (UBL4A), competes for SGTA binding. Our data show that this binding is selective, and suggest that SGTA can bind either BAG6, or UBL4A, but not both at the same time. We adapted our in vitro binding assay to study the association of BAG6 with an immobilized tail-anchored protein, Sec61β, and find both the UBL and BAG domains are dispensable for binding this substrate. This conclusion was further supported using a heterologous subcellular localization assay in yeast, where the BAG6-dependent nuclear relocalization of a second tail-anchored protein, GFP-Sed5, also required neither the UBL, nor the BAG domain of BAG6.

Significance

On the basis of these findings, we propose a working model where the large central region of the BAG6 protein provides a binding site for a diverse group of substrates, many of which expose a hydrophobic stretch of polypeptide. This arrangement would enable the BAG6 complex to bring together its substrates with potential effectors including those recruited via its N-terminal UBL. Such effectors may include SGTA, and the resulting assemblies influence the subsequent fate of the hydrophobic BAG6 substrates.     

Validation of Reliable Reference Genes for Real-Time PCR in Human Umbilical Vein Endothelial Cells on Substrates with Different Stiffness

Background

The mechanical properties of cellular microenvironments play important roles in regulating cellular functions. Studies of the molecular response of endothelial cells to alterations in substrate stiffness could shed new light on the development of cardiovascular disease. Quantitative real-time PCR is a current technique that is widely used in gene expression assessment, and its accuracy is highly dependent upon the selection of appropriate reference genes for gene expression normalization. This study aimed to evaluate and identify optimal reference genes for use in studies of the response of endothelial cells to alterations in substrate stiffness.

Methodology/Principal Findings

Four algorithms, GeNorm^PLUS, NormFinder, BestKeeper, and the Comparative ΔCt method, were employed to evaluate the expression of nine candidate genes. We observed that the stability of potential reference genes varied significantly in human umbilical vein endothelial cells on substrates with different stiffness. B2M, HPRT-1, and YWHAZ are suitable for normalization in this experimental setting. Meanwhile, we normalized the expression of YAP and CTGF using various reference genes and demonstrated that the relative quantification varied according to the reference genes.

Conclusion/Significance:

Consequently, our data show for the first time that B2M, HPRT-1, and YWHAZ are a set of stably expressed reference genes for accurate gene expression normalization in studies exploring the effect of subendothelial matrix stiffening on endothelial cell function. We furthermore caution against the use of GAPDH and ACTB for gene expression normalization in this experimental setting because of the low expression stability in this study.     

In vivo imaging of endogenous enzyme activities using luminescent 1,2-dioxetane compounds

Background

Here we present a non-invasive imaging method for visualizing endogenous enzyme activities in living animals. This optical imaging method is based on an energy transfer principle termed chemically initiated electron exchange luminescence (CIEEL). The light energy is provided by enzymatic activation of metastable 1,2-dioxetane substrates, whose protective groups are removed by hydrolytic enzymes such as β-galactosidase and alkaline phosphatase. In the presence of a nearby fluorescent recipient, the chemical energy within the activated substrate is then transferred via formation of a charge-transfer complex with the fluorophore, a mechanism closely related to glow stick chemistry.

Results

Efficient CIEEL energy transfer requires close proximity between the trigger enzyme and the fluorescent recipient. Using cells stained with fluorescent dialkylcarbocyanines as the energy recipients, we demonstrated CIEEL imaging of cellular β-galactosidase or alkaline phosphatase activity. In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.

Conclusions

In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity. In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.     

Combining Substrate Specificity Analysis with Support Vector Classifiers Reveals Feruloyl Esterase as a Phylogenetically Informative Protein Group

Background

Our understanding of how fungi evolved to develop a variety of ecological niches, is limited but of fundamental biological importance. Specifically, the evolution of enzymes affects how well species can adapt to new environmental conditions. Feruloyl esterases (FAEs) are enzymes able to hydrolyze the ester bonds linking ferulic acid to plant cell wall polysaccharides. The diversity of substrate specificities found in the FAE family shows that this family is old enough to have experienced the emergence and loss of many activities.

Methodology/Principal Findings

In this study we evaluate the relative activity of FAEs against a variety of model substrates as a novel predictive tool for Ascomycota taxonomic classification. Our approach consists of two analytical steps; (1) an initial unsupervised analysis to cluster the FAEs substrate specificity data which were generated by cultivation of 34 Ascomycota strains and then an analysis of the produced enzyme cocktail against 10 substituted cinnamate and phenylalkanoate methyl esters, (2) a second, supervised analysis for training a predictor built on these substrate activities. By applying both linear and non-linear models we were able to correctly predict the taxonomic Class (∼86% correct classification), Order (∼88% correct classification) and Family (∼88% correct classification) that the 34 Ascomycota belong to, using the activity profiles of the FAEs.

Conclusion/Significance

The good correlation with the FAEs substrate specificities that we have defined via our phylogenetic analysis not only suggests that FAEs are phylogenetically informative proteins but it is also a considerable step towards improved FAEs functional prediction.     

Fast multiclonal clusterization of V(D)J recombinations from high-throughput sequencing

Background

V(D)J recombinations in lymphocytes are essential for immunological diversity. They are also useful markers of pathologies. In leukemia, they are used to quantify the minimal residual disease during patient follow-up. However, the full breadth of lymphocyte diversity is not fully understood.

Results

We propose new algorithms that process high-throughput sequencing (HTS) data to extract unnamed V(D)J junctions and gather them into clones for quantification. This analysis is based on a seed heuristic and is fast and scalable because in the first phase, no alignment is performed with germline database sequences. The algorithms were applied to TR γ HTS data from a patient with acute lymphoblastic leukemia, and also on data simulating hypermutations. Our methods identified the main clone, as well as additional clones that were not identified with standard protocols.

Conclusions

The proposed algorithms provide new insight into the analysis of high-throughput sequencing data for leukemia, and also to the quantitative assessment of any immunological profile. The methods described here are implemented in a C++ open-source program called Vidjil.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-409) contains supplementary material, which is available to authorized users.     

Sodium Current Reduction Unmasks a Structure-Dependent Substrate for Arrhythmogenesis in the Normal Ventricles

Background

Organ-scale arrhythmogenic consequences of source-sink mismatch caused by impaired excitability remain unknown, hindering the understanding of pathophysiology in disease states like Brugada syndrome and ischemia.

Objective

We sought to determine whether sodium current (I_Na) reduction in the structurally normal heart unmasks a regionally heterogeneous substrate for the induction of sustained arrhythmia by premature ventricular contractions (PVCs).

Methods

We conducted simulations in rabbit ventricular computer models with 930 unique combinations of PVC location (10 sites) and coupling interval (250–400 ms), I_Na reduction (30 or 40% of normal levels), and post-PVC sinus rhythm (arrested or persistent). Geometric characteristics and source-sink mismatch were quantitatively analyzed by calculating ventricular wall thickness and a newly formulated 3D safety factor (SF), respectively.

Results

Reducing I_Na to 30% of its normal level created a substrate for sustained arrhythmia induction by establishing large regions of critical source-sink mismatch (SF<1) for ectopic wavefronts propagating from thin to thick tissue. In the same simulations but with 40% of normal I_Na, PVCs did not induce reentry because the volume of tissue with SF<1 was >95% smaller. Likewise, when post-PVC sinus activations were persistent instead of arrested, no ectopic excitations initiated sustained reentry because sinus activation breakthroughs engulfed the excitable gap.

Conclusion

Our new SF formulation can quantify ectopic wavefront propagation robustness in geometrically complex 3D tissue with impaired excitability. This novel methodology was applied to show that I_Na reduction precipitates source-sink mismatch, creating a potent substrate for sustained arrhythmia induction by PVCs originating near regions of ventricular wall expansion, such as the RV outflow tract.     

Deep Sequencing of Target Linkage Assay-Identified Regions in Familial Breast Cancer: Methods,Analysis Pipeline and Troubleshooting

Background

The classical candidate-gene approach has failed to identify novel breast cancer susceptibility genes. Nowadays, massive parallel sequencing technology allows the development of studies unaffordable a few years ago. However, analysis protocols are not yet sufficiently developed to extract all information from the huge amount of data obtained.

Methodology/Principal Findings

In this study, we performed high throughput sequencing in two regions located on chromosomes 3 and 6, recently identified by linkage studies by our group as candidate regions for harbouring breast cancer susceptibility genes. In order to enrich for the coding regions of all described genes located in both candidate regions, a hybrid-selection method on tiling microarrays was performed.

Conclusions/Significance

We developed an analysis pipeline based on SOAP aligner to identify candidate variants with a high real positive confirmation rate (0.89), with which we identified eight variants considered candidates for functional studies. The results suggest that the present strategy might be a valid second step for identifying high penetrance genes.     

Thyroid Hormone Reverses Aging-Induced Myocardial Fatty Acid Oxidation Defects and Improves the Response to Acutely Increased Afterload

Background

Subclinical hypothyroidism occurs during aging in humans and mice and may contribute to the development of heart failure. Aging also impairs myocardial fatty acid oxidation, causing increased reliance on flux through pyruvate dehydrogenase (PDH) to maintain function. We hypothesize that the metabolic changes in aged hearts make them less tolerant to acutely increased work and that thyroid hormone supplementation reverses these defects.

Methods

Studies were performed on young (Young, 4–6 months) and aged (Old, 22–24 months) C57/BL6 mice at standard (50 mmHg) and high afterload (80 mmHg). Another aged group received thyroid hormone for 3 weeks (Old-TH, high afterload only). Function was measured in isolated working hearts along with substrate fractional contributions (Fc) to the citric acid cycle (CAC) using perfusate with ¹³C labeled lactate, pyruvate, glucose and unlabeled palmitate and insulin.

Results

Old mice maintained cardiac function under standard workload conditions, despite a marked decrease in unlabeled (presumably palmitate) Fc and relatively similar individual carbohydrate contributions. However, old mice exhibited reduced palmitate oxidation with diastolic dysfunction exemplified by lower -dP/dT. Thyroid hormone abrogated the functional and substrate flux abnormalities in aged mice.

Conclusion

The aged heart shows diminished ability to increase cardiac work due to substrate limitations, primarily impaired fatty acid oxidation. The heart accommodates slightly by increasing efficiency through oxidation of carbohydrate substrates. Thyroid hormone supplementation in aged mice significantly improves cardiac function potentially through restoration of fatty acid oxidation.     

Crystal Structure of the S. solfataricus Archaeal Exosome Reveals Conformational Flexibility in the RNA-Binding Ring

Background

The exosome complex is an essential RNA 3′-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry.

Methodology/Principal Findings

Here we report an asymmetric 2.9 Å Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation.

Conclusion/Significance

This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.     

General Intelligence in Another Primate: Individual Differences across Cognitive Task Performance in a New World Monkey (Saguinus oedipus)

Background

Individual differences in human cognitive abilities show consistently positive correlations across diverse domains, providing the basis for the trait of “general intelligence” (g). At present, little is known about the evolution of g, in part because most comparative studies focus on rodents or on differences across higher-level taxa. What is needed, therefore, are experiments targeting nonhuman primates, focusing on individual differences within a single species, using a broad battery of tasks. To this end, we administered a large battery of tasks, representing a broad range of cognitive domains, to a population of captive cotton-top tamarin monkeys (Saguinus oedipus).

Methodology and Results

Using a Bayesian latent variable model, we show that the pattern of correlations among tasks is consistent with the existence of a general factor accounting for a small but significant proportion of the variance in each task (the lower bounds of 95% Bayesian credibility intervals for correlations between g and task performance all exceed 0.12).

Conclusion

Individual differences in cognitive abilities within at least one other primate species can be characterized by a general intelligence factor, supporting the hypothesis that important aspects of human cognitive function most likely evolved from ancient neural substrates.     

Linking ClinicalTrials.gov and PubMed to Track Results of Interventional Human Clinical Trials

Objective

In an effort to understand how results of human clinical trials are made public, we analyze a large set of clinical trials registered at ClinicalTrials.gov, the world’s largest clinical trial registry.

Materials and Methods

We considered two trial result artifacts: (1) existence of a trial result journal article that is formally linked to a registered trial or (2) the deposition of a trial’s basic summary results within the registry.

Results

The study sample consisted of 8907 completed, interventional, phase 2-or-higher clinical trials that were completed in 2006-2009. The majority of trials (72.2%) had no structured trial-article link present. A total of 2367 trials (26.6%) deposited basic summary results within the registry. Of those , 969 trials (10.9%) were classified as trials with extended results and 1398 trials (15.7%) were classified as trials with only required basic results. The majority of the trials (54.8%) had no evidence of results, based on either linked result articles or basic summary results (silent trials), while a minimal number (9.2%) report results through both registry deposition and publication.

Discussion

Our study analyzes the body of linked knowledge around clinical trials (which we refer to as the “trialome”). Our results show that most trials do not report results and, for those that do, there is minimal overlap in the types of reporting. We identify several mechanisms by which the linkages between trials and their published results can be increased.

Conclusion

Our study shows that even when combining publications and registry results, and despite availability of several information channels, trial sponsors do not sufficiently meet the mandate to inform the public either via a linked result publication or basic results submission.     

Mitochondrial Targeted Coenzyme Q,Superoxide, and Fuel Selectivity in Endothelial Cells

Background

Previously, we reported that the “antioxidant” compound “mitoQ” (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates.

Methods and Results

To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity.

Conclusions

In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over fatty acid oxidation at the intact cell level.     

Genome Scan for Locus Involved in Mandibular Prognathism in Pedigrees from China

Background

It is well known that genetic components play an important role in the etiology of mandibular prognathism, but few susceptibility loci have been mapped.

Methodology

In order to identify linkage regions for mandibular prognathism, we analyzed two Chinese pedigrees with 6,090 genome-wide single-nucleotide polymorphism (SNP) markers from Illumina Linkage-12 DNA Analysis Kit (average spacing 0.58 cM). Multipoint parametric and non-parametric (model-free) linkage analyses were used for the pedigrees.

Principal Finding

The most statistically significant linkage results were with markers on chromosome 4 (LOD  = 3.166 and NPL = 3.65 with rs 875864, 4p16.1, 8.38 cM). Candidate genes within the 4p16.1 include EVC, EVC2.

Conclusion

We detected a novel suggestive linkage locus for mandibular prognathism in two Chinese pedigrees, and this linkage region provides target for susceptibility gene identification, a process that will provide important insights into the molecular and cellular basis of mandibular prognathism.     

Study on measuring device arrangement of array-type CdTe detector for BNCT-SPECT

Aim

To design the measuring device arrangement of array-type CdTe detector for BNCT-SPECT.

Background

In a boron neutron capture therapy, a very serious unsolved problem exists, namely that the treatment effect for BNCT cannot be known during irradiation in real time. Therefore, we have been developing a so-called BNCT-SPECT with a CdTe detector, which can obtain a three-dimensional image for the BNCT treatment effect by measuring 478 keV gamma-rays emitted from the excited state of ⁷Li nucleus created by the ¹⁰B(n,α) reaction. However, no practical uses were realized at present, because BNCT-SPECT requires very severe conditions for spatial resolution, measuring time, statistical accuracy and energy resolution.

Materials and methods

The design study was performed with numerical simulations carried out by a 3-dimenaional transport code, MCNP5 considering the detector assembly, irradiation room and even arrangement of arrayed CdTe crystals.

Results

The estimated count rate of 478 keV gamma-rays was sufficiently large being more than the target value of over 1000 counts/h. However, the S/N ratio did not meet the target of S/N > 1. We confirmed that deterioration of the S/N ratio was caused by the influence of Compton scattering especially due to capture gamma-rays of hydrogen. Theoretical calculations were thereafter carried out to find out whether anti-Compton measurement in an array-type CdTe detector could decrease the noise due to Compton scatterings.

Conclusions

The calculation result showed that the anti-coincidence would possibly increase the S/N ratio. In the next phase, an arrayed detector with two CdTe crystals will be produced to test removal possibility of the anti-coincident event.     

BCL10GFP fusion protein as a substrate for analysis of determinants required for Mucosa-Associated Lymphoid Tissue 1 (MALT1)-mediated cleavage

Background

MALT1 belongs to a family of paracaspase and modulates NF-κB signaling pathways through its scaffolding function and proteolytic activity. MALT1 cleaves protein substrates after a positively charged Arginine residue. BCL10, a 233 amino acids polypeptide, is identified as one of the MALT1 proteolytic substrates. MALT1 cleaves BCL10 at the C-terminal end of Arg228. A mere 5 amino acids difference between the substrate and the proteolytic product made it difficult to tell whether the cleavage event took place by using a simple western blot analysis. Here, BCL10GFP was constructed and utilized to examine the specificity and domain determinants for MALT1 cleavage in cells.

Methods

Various BCL10GFP constructs were transfected into HEK293T cell with MALT1 construct by using calcium phosphate-DNA precipitation method. Lysates of transfectants were resolved by SDS/PAGE and analyzed by western blot analysis.

Results

BCL10GFP was proteolytically processed by MALT1 as BCL10. The integrity of caspase recruitment domain (CARD) and MALT1-interacting domain on BCL10 were required for MALT1 proteolytic activity. Besides the invariant P1 cleavage site Arg228, P4 Leu225 played a role in defining BCL10 as a good substrate for MALT1.

Conclusions

We offered a way of monitoring the catalytic activity of MALT1 in HEK293T cells using BCL10GFP as a substrate. BCL10GFP can be utilized as a convenient tool for studying the determinants for efficient MALT1 cleavage in HEK293T cells