Proteoglycanomics: tools to unravel the biological function of glycosaminoglycans

Glycosylation is the most frequent PTM and contributes significantly to the function of proteins depending on the type of glycosylation. Especially glycan structures like the glycosaminoglycans are considered to constitute themselves the major function of the glycoconjugate which is therefore termed proteoglycan. Here we review recent views on and novel tools for analysing the proteoglycanome, which are directly related to the type of glycanation under investigation. We define the major function of the proteoglycanome to be its interaction with various proteins in many different (patho-)physiological conditions. This is exemplified by the differential glycosaminoglycan-interactome of healthy versus arthritic patient sera.     

Mutants as tools to understand cellular and molecular drought tolerance mechanisms

The use of mutants: a most promising way to detect genes involved in development or in response to environmental stress. The model species Arabidopsis, particularly amenable to dissect the genetics and molecular mechanisms underlying physiological responses, also offers the advantage of a wide variety of mutants. As far as drought tolerance is concerned, hormonal mutants, impaired in hormone biosynthesis — deficient mutants — or in the signal transduction pathway—responsive mutants—provide a valuable tool to analyse the role of phytohormone interaction in the plant drought behaviour as well as to differentiate the mutant phenotypes with new criteria.These two categories of mutants (in particular the abscisic acid, ABA, mutants) were shown to be affected in developmental processes during seed maturation-in the desiccation phase- and/or in response to environmental stress (drought, ...) in vegetative tissues. The present report will focus on this last aspect: alterations in drought responses in vegetative tissues (adaptive strategies and drought tolerance mechanisms) essentially in Arabidopsis hormonal mutants (ABA-deficient and ABA-insensitive, GA-deficient, auxin and ethylene-insensitive).Some of the results are discussed with regard to the predicted functions of genes affected by the mutations.     

Developing tools to unravel the biological secrets of Rosellinia necatrix, an emergent threat to woody crops

White root rot caused by Rosellinia necatrix is one of the most destructive diseases of many woody plants in the temperate regions of the world, particularly in Europe and Asia. Recent outbreaks of R. necatrix around the globe have increased the interest in this pathogen. Although the ecology of the disease has been poorly studied, recent genetic and molecular advances have opened the way for future detailed studies of this fungus. TAXONOMY: Rosellinia necatrix Prilleux. Kingdom Fungi; subdivision Ascomycotina; class Euascomycetes; subclass Pyrenomycetes; order Sphaeriales, syn. Xylariales; family Xylariaceae; genus Rosellinia. IDENTIFICATION: Fungal mycelium is present on root surfaces and under the bark, forming mycelium fans, strands or cords. A typical presence of pear-shaped or pyriform swellings can be found above the hyphal septum (with diameters of up to 13 μm). Sclerotia are black, hard and spherical nodules, several millimetres in diameter. Black sclerotia crusts may also form on roots. On synthetic media, it forms microsclerotia: irregular rough bodies composed of a compact mass of melanized, interwoven hyphae with no differentiated cells. Chlamydospores are almost spherical (15 μm in diameter). Synnemata, also named coremia (0.5-1.5 mm in length), can be formed from sclerotia or from mycelial masses. Conidia (3-5 μm in length and 2.5-3 μm in width) are very difficult to germinate in vitro. Ascospores are monostichous, situated inside a cylindrical, long-stalked ascus. They are ellipsoidal and cymbiform (36-46 μm in length and 5.5-6.3 μm in width). HOST RANGE: This fungus can attack above 170 different plant hosts from 63 genera and 30 different families, including vascular plants and algae. Some are of significant economic importance, such as Coffea spp., Malus spp., Olea europaea L., Persea americana Mill., Prunus spp. and Vitis vinifera L. DISEASE SYMPTOMS: Rosellinia necatrix causes white (or Dematophora) root rot, which, by aerial symptoms, shows a progressive weakening of the plant, accompanied by a decline in vigour. The leaves wilt and dry, and the tree can eventually die. White cottony mycelium and mycelial strands can be observed in the crown and on the root surface. On woody plant roots, the fungus can be located between the bark and the wood, developing typical mycelium fans, invading the whole root and causing general rotting. DISEASE CONTROL: Some approaches have been attempted involving the use of tolerant plants and physical control (solarization). Chemical control in the field and biological control methods are still under development.     

Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes

Rearrangements of our genome can be responsible for inherited as well as sporadic traits. The analyses of chromosome breakpoints in the proximal short arm of Chromosome 17 (17p) reveal nonallelic homologous recombination (NAHR) as a major mechanism for recurrent rearrangements whereas nonhomologous end-joining (NHEJ) can be responsible for many of the nonrecurrent rearrangements. Genome architectural features consisting of low-copy repeats (LCRs), or segmental duplications, can stimulate and mediate NAHR, and there are hotspots for the crossovers within the LCRs. Rearrangements introduce variation into our genome for selection to act upon and as such serve an evolutionary function analogous to base pair changes. Genomic rearrangements may cause Mendelian diseases, produce complex traits such as behaviors, or represent benign polymorphic changes. The mechanisms by which rearrangements convey phenotypes are diverse and include gene dosage, gene interruption, generation of a fusion gene, position effects, unmasking of recessive coding region mutations (single nucleotide polymorphisms, SNPs, in coding DNA) or other functional SNPs, and perhaps by effects on transvection.     

Caveolae--from ultrastructure to molecular mechanisms

Almost 50 years after the first sighting of small pits that covered the surface of mammalian cells, investigators are now getting to grips with the detailed workings of these enigmatic structures that we now know as caveolae.     

Primary immune deficiencies unravel the molecular basis of immune response

Primary immune deficiencies (PID) represent inborn errors of immunity. Over the years, detailed analysis of the clinical and laboratory features associated with these unique and rare disorders have shed light on the complex array of signals and processes that govern development and activation of the immune system. While the first examples of PID pertained to severe defects in lymphoid development, more recently a variety of gene defects have been identified in humans that do not compromize the ability to generate lymphocytes, but rather result in profound immune dysregulation. In many cases, identification of the molecular and cellular bases of PID has preceeded development of animal models by gene targeting. Finally, since the very first cases reported in humans, PID have also represented a unique tool to investigate the efficacy of novel therapeutic approaches (from molecular therapy to hematopoietic stem cell transplantation to somatic cells gene therapy), that have been applied or may apply to a variety of more common human diseases.     

Prediction of drug resistance in M. tuberculosis: molecular mechanisms,tools, and applications

Management of Tuberculosis is complicated by the emergence of drug resistant strains of Mycobacterium tuberculosis and this poses a threat to the success of Tuberculosis control programmes. Drug susceptibility testing by culture is time-consuming and technically difficult. It is known that resistance to drugs is due to a number of genomic mutations in specific genes of M. tuberculosis. These mutations in combination with molecular techniques can be used as markers for drug resistance, since drug susceptible isolates lack the corresponding gene mutations. This review focuses on molecular mechanisms, methods and applications as a possible new diagnostic tool for the early molecular detection of drug resistance in M. tuberculosis.     

Software tools for molecular microscopy

In our role as the editors of a special edition of the Journal of Structural Biology published in 1996 and devoted to the development of software tools, we offer our view of past developments and future prospects in this area. The astonishing progress in computer hardware over the past decade has fueled a significant increase in computational power available for the solution of macromolecular structures. At the same time the relatively slow growth and development of the accompanying software reflects the difficulties of developing large, complex and very specialized analytical methods.     

Monoaminergic mechanisms in affective disorders

The monoamine hypothesis of depression originally proposed that depression is caused by a central deficiency of biogenic amines, and antidepressants were considered to work by correcting this deficiency. In the course of time, many studies have analysed monoamine metabolites in the urine, plasma and cerebrospinal fluid of patients and healthy controls under different conditions to test the hypothesis. These studies have failed to identify a robust metabolic disorder in depressive patients as a group. Certain subgroups of depressed patients have shown deviations in biogenic amine metabolism, the most consistent being reduced levels of the major serotonin and dopamine metabolites in the cerebrospinal fluid. Noradrenaline metabolism is influenced by the activity of the sympathetic nervous system, and thus increases in anxious patients regardless of their clinical diagnosis. On the other hand, development of new antidepressants and advances in receptor techniques, together with modern electrophysiologic and behavioural studies have given increasing support to a receptor supersensitivity hypothesis of depression, based on the evidence that antidepressants lead to subsensitivity or down regulation of beta-adrenoceptors, and adaptive changes may occur also in other receptor systems after two three weeks of antidepressant treatment. There is also growing evidence on the manifold interplay of noradrenergic and serotonergic systems in the mechanism of actions of effective antidepressant treatments, including the new and more selective therapeutic compounds. The rapidly increasing knowledge of the neurotransmitter receptors as well as of the relations between the different regulatory systems may lead to more specific intervention strategies in efforts to correct the biological malfunction in the heterogeneous collection of diseases classified as affective disorders.     

GPU-powered tools boost molecular visualization

Recent advances in experimental structure determination provide a wealth of structural data on huge macromolecular assemblies such as the ribosome or viral capsids, available in public databases. Further structural models arise from reconstructions using symmetry orders or fitting crystal structures into low-resolution maps obtained by electron-microscopy or small angle X-ray scattering experiments. Visual inspection of these huge structures remains an important way of unravelling some of their secrets. However, such visualization cannot conveniently be carried out using conventional rendering approaches, either due to performance limitations or due to lack of realism. Recent developments, in particular drawing benefit from the capabilities of Graphics Processing Units (GPUs), herald the next generation of molecular visualization solutions addressing these issues. In this article, we present advances in computer science and visualization that help biologists visualize, understand and manipulate large and complex molecular systems, introducing concepts that remain little-known in the bioinformatics field. Furthermore, we compile currently available software and methods enhancing the shape perception of such macromolecular assemblies, for example based on surface simplification or lighting ameliorations.     

Novel NMR tools to study structure and dynamics of biomembranes

Nuclear magnetic resonance (NMR) studies on biomembranes have benefited greatly from introduction of magic angle spinning (MAS) NMR techniques. Improvements in MAS probe technology, combined with the higher magnetic field strength of modern instruments, enables almost liquid-like resolution of lipid resonances. The cross-relaxation rates measured by nuclear Overhauser enhancement spectroscopy (NOESY) provide new insights into conformation and dynamics of lipids with atomic-scale resolution. The data reflect the tremendous motional disorder in the lipid matrix. Transfer of magnetization by spin diffusion along the proton network of lipids is of secondary relevance, even at a long NOESY mixing time of 300 ms. MAS experiments with re-coupling of anisotropic interactions, like the 13C-(1)H dipolar couplings, benefit from the excellent resolution of 13C shifts that enables assignment of the couplings to specific carbon atoms. The traditional 2H NMR experiments on deuterated lipids have higher sensitivity when conducted on oriented samples at higher magnetic field strength. A very large number of NMR parameters from lipid bilayers is now accessible, providing information about conformation and dynamics for every lipid segment. The NMR methods have the sensitivity and resolution to study lipid-protein interaction, lateral lipid organization, and the location of solvents and drugs in the lipid matrix.     

High-resolution proteomics unravel architecture and molecular diversity of native AMPA receptor complexes

AMPA-type glutamate receptors (AMPARs) are responsible for a variety of processes in the mammalian brain including fast excitatory neurotransmission, postsynaptic plasticity, or synapse development. Here, with comprehensive and quantitative proteomic analyses, we demonstrate that native AMPARs are macromolecular complexes with a large molecular diversity. This diversity results from coassembly of the known AMPAR subunits, pore-forming GluA and three types of auxiliary proteins, with 21 additional constituents, mostly secreted proteins or transmembrane proteins of different classes. Their integration at distinct abundance and stability establishes the heteromultimeric architecture of native AMPAR complexes: a defined core with a variable periphery resulting in an apparent molecular mass between 0.6 and 1 MDa. The additional constituents change the gating properties of AMPARs and provide links to the protein dynamics fundamental for the complex role of AMPARs in formation and operation of glutamatergic synapses.