Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of the Performance of Information Theory-Based Methods and Cross-Correlation to Estimate the Functional Connectivity in Cortical Networks

Functional connectivity of in vitro neuronal networks was estimated by applying different statistical algorithms on data collected by Micro-Electrode Arrays (MEAs). First we tested these “connectivity methods” on neuronal network models at an increasing level of complexity and evaluated the performance in terms of ROC (Receiver Operating Characteristic) and PPC (Positive Precision Curve), a new defined complementary method specifically developed for functional links identification. Then, the algorithms better estimated the actual connectivity of the network models, were used to extract functional connectivity from cultured cortical networks coupled to MEAs. Among the proposed approaches, Transfer Entropy and Joint-Entropy showed the best results suggesting those methods as good candidates to extract functional links in actual neuronal networks from multi-site recordings.     

Site-directed mutagenesis of two aromatic residues lining the active site pocket of the yeast Ltp1

We mutated Trp(134) and Tyr(135) of the yeast LMW-PTP to explore their catalytic roles, demonstrating that the mutations of Trp(134) to Tyr or Ala, and Tyr(135) to Ala, all interfere with the formation of the phosphorylenzyme intermediate, a phenomenon that can be seen by the decrease in the kinetic constant of the chemical step (k(3)). Furthermore, we noted that the Trp(134) to Ala mutation causes a dramatic drop in k(cat)/K(m) and a slight enhancement of the dissociation constant K(s). The conservative mutant W134Y shows a k(cat)/K(m) very close to that of wild type, probably compensating the two-fold decrease of k(3) with an increase in substrate affinity. The Y135A mutation enhances the substrate affinity, but reduces the enzyme phosphorylation rate. The replacement of Trp(134) with alanine interferes with the partition between phosphorylenzyme hydrolysis and phosphotransfer from the phosphorylenzyme to glycerol and abolish the enzyme activation by adenine. Finally, we found that mutation of Trp(134) to Ala causes a dramatic change in the pH-rate profile that becomes similar to that of the D132A mutant, suggesting that an aromatic residue in position 134 is necessary to assist the proper positioning of the proton donor in the transition state of the chemical step.     

CXC chemokine receptor 4 is expressed in uveal malignant melanoma and correlates with the epithelioid-mixed cell type

PURPOSE: Although relatively rare, uveal melanoma is the most common ocular tumor of adults. Up to half of uveal melanoma patients die of metastatic disease. CXCR4, a chemokine receptor, is a prognostic factor in cutaneous melanoma involved in angiogenesis and metastasis formation. The aim of this study was to evaluate the expression of CXCR4 in uveal melanoma. METHODS: CXCR4 was detected by immunohistochemistry in 44 samples of uveal melanoma. Staining was categorized into three semiquantitative classes based on the rate of stained (positive) tumor cells: absence of staining, <50% of cell (+) and >50% (++). Correlations between CXCR4 expression, data on patient and tumor features were studied by contingency tables and the chi2 test. Time-to-event curves were studied using the Kaplan-Meier method. Univariate analysis was performed using the log-rank test. Ninety-five percent confidence intervals (95% CI) of hazard ratios were also reported. RESULTS: Staining for CXCR4 protein was absent in 18 tumors (40.9%), present in <50% of cells in 19 (43.2%) and in >50% of cells in 7 (15.9%) tumors. CXCR4 expression correlated to the epithelioid-mixed cell type (P=0.030). No statistically significant relation emerged between CXCR4 expression, largest tumor diameter (LTD) and extracellular matrix patterns as evaluated through histological patterns stained with periodic acid-Schiff (PAS). Events occurred in 2 out of 18 patients (11.1%) with negative tumors (2 deaths), in 3 out of 19 patients (15.8%) with <50% of positive tumor cells (2 deaths and 1 occurrence of metastases) and in 1 out of 7 patients (14.3%) with >50% of positive tumor cells (1 occurrence of metastases). The cell type (P=0.0457) but not CXCR4 showed prognostic value at univariate analysis. CONCLUSION: This study shows that CXCR4 is commonly expressed in uveal melanoma and correlates with cell type a well-established prognostic factor.     

Coralloid root regeneration on Macrozamia megagametophites

Abstract

Megagametophytes of Macrozamia communis were incubated in White's Basal Medium and in White's Basal Medium modified with 2,4-D and kinetin. On the medium enriched with growth substances, regeneration of coralloid roots was induced. These are morphologically identical to sporophytic coralloid roots, without any endosymbiont and displaying negative geotropism. These results confirm the fact that coralloid roots represent an inherent feature of the root system of the Cycadales rather than being the result of induction by microbial factors. Therefore it is possible to suggest that coralloid roots represent vestigial pneumatophores.     

A GFP-based system to uncouple mRNA transport from translation in a single living neuron

An inducible fluorescent system based on GFP is presented that allows for the uncoupling of dendritic mRNA transport from subsequent protein synthesis at the single cell level. The iron-responsive element (IRE) derived from ferritin mRNA in the 5'-UTR of the GFP reporter mRNA renders translation of its mRNA dependent on iron. The addition of the full-length 3'-UTR of the Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) after the stop codon of the GFP reading frame targets the reporter mRNA to dendrites of transfected fully polarized hippocampal neurons. As we show by time-lapse videomicroscopy, iron specifically turns on GFP reporter protein synthesis in a single transfected hippocampal neuron. We investigate whether GFP expression is affected--in addition to iron--by synaptic activity. Interestingly, synaptic activity has a clear stimulatory effect. Most importantly, however, this activity-dependent protein synthesis is critically dependent on the presence of the full-length 3'-UTR of CaMKIIalpha confirming that this sequence contains translational activation signals. The IRE-based system represents a new convenient tool to study local protein synthesis in mammalian cells where mRNA localization to a specific intracellular compartment occurs.     

Implementation of a molecular epidemiology approach to human pleural malignant mesothelioma

The carcinogenic effect of asbestos has been reported in the literature since 40 years, and early studies describing the epidemic occurrence of malignant mesothelioma (MM) in asbestos workers, have become a paradigm of occupational cancer research. Research on MM was abandoned for many years since MM was considered as an asbestos-related disease, interesting only from a perspective of disease control and preventive policies. The introduction of new biological endpoints in the epidemiological studies has boosted research in the field, providing new tools for the study of emerging priority in cancer research and in public health. This approach, known as molecular epidemiology has a great potential in the study of MM, contributing to the understanding of susceptibility factors, to the evaluation of cancer risk in people occupationally or environmentally exposed to carcinogens, and to the enhancement of diagnosis and therapy. A comprehensive approach based on the use of banks of biological samples is presented and its advantages discussed here. The application of innovative endpoints, such as oncoproteins in biologic fluids, genetic polimorphisms, or gene function is discussed, and relevant literature reviewed.     

Exploring the concept of interaction computing through the discrete algebraic analysis of the Belousov–Zhabotinsky reaction

Interaction computing (IC) aims to map the properties of integrable low-dimensional non-linear dynamical systems to the discrete domain of finite-state automata in an attempt to reproduce in software the self-organizing and dynamically stable properties of sub-cellular biochemical systems. As the work reported in this paper is still at the early stages of theory development it focuses on the analysis of a particularly simple chemical oscillator, the Belousov–Zhabotinsky (BZ) reaction. After retracing the rationale for IC developed over the past several years from the physical, biological, mathematical, and computer science points of view, the paper presents an elementary discussion of the Krohn–Rhodes decomposition of finite-state automata, including the holonomy decomposition of a simple automaton, and of its interpretation as an abstract positional number system. The method is then applied to the analysis of the algebraic properties of discrete finite-state automata derived from a simplified Petri net model of the BZ reaction. In the simplest possible and symmetrical case the corresponding automaton is, not surprisingly, found to contain exclusively cyclic groups. In a second, asymmetrical case, the decomposition is much more complex and includes five different simple non-abelian groups whose potential relevance arises from their ability to encode functionally complete algebras. The possible computational relevance of these findings is discussed and possible conclusions are drawn.