Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins

Background

Scaffolding proteins of the intersectin (ITSN) family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs.

Methodology/Principal Findings

We have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform.

Conclusions/Significance

Our results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.     

Classification of large microarray datasets using fast random forest construction

Random forest is an ensemble classification algorithm. It performs well when most predictive variables are noisy and can be used when the number of variables is much larger than the number of observations. The use of bootstrap samples and restricted subsets of attributes makes it more powerful than simple ensembles of trees. The main advantage of a random forest classifier is its explanatory power: it measures variable importance or impact of each factor on a predicted class label. These characteristics make the algorithm ideal for microarray data. It was shown to build models with high accuracy when tested on high-dimensional microarray datasets. Current implementations of random forest in the machine learning and statistics community, however, limit its usability for mining over large datasets, as they require that the entire dataset remains permanently in memory. We propose a new framework, an optimized implementation of a random forest classifier, which addresses specific properties of microarray data, takes computational complexity of a decision tree algorithm into consideration, and shows excellent computing performance while preserving predictive accuracy. The implementation is based on reducing overlapping computations and eliminating dependency on the size of main memory. The implementation's excellent computational performance makes the algorithm useful for interactive data analyses and data mining.     

Enterohemorrhagic Escherichia coli infection stimulates Shiga toxin 1 macropinocytosis and transcytosis across intestinal epithelial cells

Gastrointestinal infection with Shiga toxins producing enterohemorrhagic Escherichia coli causes the spectrum of gastrointestinal and systemic complications, including hemorrhagic colitis and hemolytic uremic syndrome, which is fatal in ～10% of patients. However, the molecular mechanisms of Stx endocytosis by enterocytes and the toxins cross the intestinal epithelium are largely uncharacterized. We have studied Shiga toxin 1 entry into enterohemorrhagic E. coli-infected intestinal epithelial cells and found that bacteria stimulate Shiga toxin 1 macropinocytosis through actin remodeling. This enterohemorrhagic E. coli-caused macropinocytosis occurs through a nonmuscle myosin II and cell division control 42 (Cdc42)-dependent mechanism. Macropinocytosis of Shiga toxin 1 is followed by its transcytosis to the basolateral environment, a step that is necessary for its systemic spread. Inhibition of Shiga toxin 1 macropinocytosis significantly decreases toxin uptake by intestinal epithelial cells and in this way provides an attractive, antibiotic-independent strategy for prevention of the harmful consequences of enterohemorrhagic E. coli infection.     

Amyloid features and neuronal toxicity of mature prion fibrils are highly sensitive to high pressure

Prion proteins (PrP) can aggregate into toxic and possibly infectious amyloid fibrils. This particular macrostructure confers on them an extreme and still unexplained stability. To provide mechanistic insights into this self-assembly process, we used high pressure as a thermodynamic tool for perturbing the structure of mature amyloid fibrils that were prepared from recombinant full-length mouse PrP. Application of high pressure led to irreversible loss of several specific amyloid features, such as thioflavin T and 8-anilino-1-naphthalene sulfonate binding, alteration of the characteristic proteinase K digestion pattern, and a significant decrease in the β-sheet structure and cytotoxicity of amyloid fibrils. Partial disaggregation of the mature fibrils into monomeric soluble PrP was observed. The remaining amyloid fibrils underwent a change in secondary structure that led to morphologically different fibrils composed of a reduced number of proto-filaments. The kinetics of these reactions was studied by recording the pressure-induced dissociation of thioflavin T from the amyloid fibrils. Analysis of the pressure and temperature dependence of the relaxation rates revealed partly unstructured and hydrated kinetic transition states and highlighted the importance of collapsing and hydrating inter- and intramolecular cavities to overcome the high free energy barrier that stabilizes amyloid fibrils.     

Expression of recombinant actin 5C from Drosophila in the methylotrophic yeast Pichia pastoris

A system for actin expression in cells of yeast Pichia pastoris was constructed. Drosophila actin 5C, by 90% homologous to beta-actin of higher eukaryotes, was used as a target protein. To improve the procedures of target protein biosynthesis in yeast cells and of extraction and purification of recombinant actin the fusion protein GFP-actin 5C, having fluorescence protein GFP as a reporter part, was expressed and purified. The dimensions and resistance of yeast cells producing recombinant actin were characterized. It was shown that the size and form of cells depended on the accumulation of recombinant protein. The purified fusion protein was used for obtaining polyclonal antibody for testing recombinant actin.     

Changes in the mitochondrial permeability in medullary cardiovascular neurons influence the hemodynamics in rats

In acute experiments on anesthetized rats, we studied the effects of modulation of the mitochondrial permeability in medullary cardiovascular neurons (nucl. tractus solitarii, NTS, nucl. ambiguus, AMB, paramedian reticular nucleus, PMn, and lateral reticular nucleus, LRN) on the systemic arterial pressure (SAP). We were the first to show that the mitochondrial permeability is essential for medullary cardiovascular control. An increase in the mitochondrial permeability with injections of an inductor of mitochondrial transition pore opening, phenylarsine oxide (PAO, 0.5 to 504 nmol), into the medullary nuclei resulted in long-lasting decreases in the SAP; at high doses of PAO, these drops could be irreversible and led to the animal’s death. Injections of an inhibitor of mitochondrial transition pore opening, melatonin (0.7 to 70.0 nmol), into the medullary nuclei induced dose-dependent increases in the SAP. Melatonin and L-arginine were shown to demonstrate neuroprotective effects due to their ability to attenuate the consequences of increased mitochondrial permeability in medullary cardiovascular neurons. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 392–395, July–October, 2007.     

Melanocyte differentiation antigen RAB38/NY-MEL-1 induces frequent antibody responses exclusively in melanoma patients

Expression pattern and immunogenicity are critical issues that define tumor antigens as diagnostic markers and potential targets for immunotherapy. The development of SEREX (serological analysis of recombinant expression libraries) has provided substantial progress in the identification of tumor antigens eliciting both cellular and humoral immune responses in cancer patients. By SEREX, we have previously identified RAB38/NY-MEL-1 as a melanocyte differentiation antigen that is highly expressed in normal melanocytes and melanoma tissues but not in other normal tissues or cancer types. In this study, we further demonstrate that RAB38/NY-MEL-1 is strongly immunogenic, leading to spontaneous antibody responses in a significant proportion of melanoma patients. The immune response occurs solely in malignant melanoma patients and was not detected in patients with other diseases, such as vitiligo, affecting melanocytes. Fine analysis of the spontaneous anti-RAB38/NY-MEL-1 antibody response reveals a polyclonal B cell recognition targeting various epitopes, although a dominant immunogenic region was preferentially recognized. Interestingly, our data indicate that this recognition is not rigid in the course of a patient’s response, as the dominant epitope changes during the disease evolution. Implications for the understanding of spontaneous humoral immune responses are discussed.Alfred Zippelius and Asma Gati contributed equally to this work.     

Linking two worlds in polymer chemistry: The influence of block uniformity and dispersity in amphiphilic block copolypeptoids on their self-assembly

The self-assembly of block copolymers has captured the interest of scientists for many decades because it can induce ordered structures and help to imitate complex structures found in nature. In contrast to proteins, nature's most functional hierarchical structures, conventional polymers are disperse in their length distribution. Here, we synthesized hydrophilic and hydrophobic polypeptoids via solid-phase synthesis (uniform) and ring-opening polymerization (disperse). Differential scanning calorimetry measurements showed that the uniform hydrophobic peptoids converge to a maximum of the melting temperature at a much lower chain length than their disperse analogs, showing that not only the chain length but also the dispersity has a considerable impact on the thermal properties of those homopolymers. These homopolymers were then coupled to yield amphiphilic block copolypeptoids. SAXS and AFM measurements confirm that the dispersity plays a major role in microphase separation of these macromolecules, and it appears that uniform hydrophobic blocks form more ordered structures.     

Impact of Uncertainties in Exposure Assessment on Estimates of Thyroid Cancer Risk among Ukrainian Children and Adolescents Exposed from the Chernobyl Accident

The 1986 accident at the Chernobyl nuclear power plant remains the most serious nuclear accident in history, and excess thyroid cancers, particularly among those exposed to releases of iodine-131 remain the best-documented sequelae. Failure to take dose-measurement error into account can lead to bias in assessments of dose-response slope. Although risks in the Ukrainian-US thyroid screening study have been previously evaluated, errors in dose assessments have not been addressed hitherto. Dose-response patterns were examined in a thyroid screening prevalence cohort of 13,127 persons aged <18 at the time of the accident who were resident in the most radioactively contaminated regions of Ukraine. We extended earlier analyses in this cohort by adjusting for dose error in the recently developed TD-10 dosimetry. Three methods of statistical correction, via two types of regression calibration, and Monte Carlo maximum-likelihood, were applied to the doses that can be derived from the ratio of thyroid activity to thyroid mass. The two components that make up this ratio have different types of error, Berkson error for thyroid mass and classical error for thyroid activity. The first regression-calibration method yielded estimates of excess odds ratio of 5.78 Gy⁻¹ (95% CI 1.92, 27.04), about 7% higher than estimates unadjusted for dose error. The second regression-calibration method gave an excess odds ratio of 4.78 Gy⁻¹ (95% CI 1.64, 19.69), about 11% lower than unadjusted analysis. The Monte Carlo maximum-likelihood method produced an excess odds ratio of 4.93 Gy⁻¹ (95% CI 1.67, 19.90), about 8% lower than unadjusted analysis. There are borderline-significant (p = 0.101–0.112) indications of downward curvature in the dose response, allowing for which nearly doubled the low-dose linear coefficient. In conclusion, dose-error adjustment has comparatively modest effects on regression parameters, a consequence of the relatively small errors, of a mixture of Berkson and classical form, associated with thyroid dose assessment.