Spermatogonial stem cells

The mammalian seminiferous epithelium consists of a highly complex yet well-organized cell population, with germ cells in mitosis and meiosis and postmeiotic cells undergoing transformation to become spermatozoa. To study the factors which control renewal and differentiation of spermatogonial stem cells, animal models are now available which allow for arrest and restart of spermatogonial differentiation. In addition, marked progress has been made in understanding the control of apoptosis and its role in spermatogonia. For the future, spermatogonial stem cell transplantation may have important practical applications.     

Tubulin structure: insights into microtubule properties and functions

The structure of tubulin has recently been determined by electron crystallography, paving the way for a clearer understandin of the unique properties of tubulin that allow its varied functions within the cell. Some of the ongoing work on tubulin can be interpreted in terms of its structure, which can serve to guide future studies.     

Insights into the mechanisms of homologous recombination from the structure of RuvA

The recent structure determination of RuvA has provided the first insights into the structural basis for its interaction with Holliday junction DNA. Multiple copies of a helix-hairpin-helix motif which line the four grooves between the monomers in the tetrameric structure are thought to be involved in the interaction of the protein with its DNA target. This suggests that the four arms of the junction are held by RuvA in a fourfold symmetric arrangement and has fuelled ideas on the way in which components of the Ruv complex combine to catalyse the process of homologous recombination     

Protein—drug complexes important for immunoregulation and organ transplantation

Recently, two structures of the Ser/Thr phosphorylase calcineurin in complex with FK506 and its cognate immunophilin, FKBP12, have been reported, both solved by small pharmaceutical companies focused on structure-based drug design. A realization, however, that the toxicities associated with calcineurin-mediated immunosuppressants might be mechanism based has driven the current interest in alternative approaches to autoimmunity prophylaxis and preventing transplant rejection. Regulatory approval in 1995 of the immunosuppressant prodrug mycophenolate mofetil, whose active metabolite, mycophenolic acid, inhibits inosine monophosphate dehydrogenase, has focused attention on the potential significance of the de novo purine-biosynthesis pathway as a target for immunosuppressive drugs, leading ultimately to the solution of enzyme structure as a drug design target. As this and other clinically relevant targets are discovered, elaborated and refined via the activity of their cognate agents (as was the case for the phosphatase calcineurin via the activity of cyclosporin), a critical opportunity should ensue for structural biology to exert a profound effect on the future development of these therapies.     

Structural and mechanistic studies of enolase

The high-resolutionstructure of yeast enolase cocrystallized with its equilibrium mixture of substrate and product reveals the stereochemistry of substrate/product binding and therefore the groups responsible for acid/base catalysis and stabilization of the enolate intermediate. Expression and characterization of site-specific mutant forms of the enzyme have confirmed the roles of amino acid side chains in the catalysis of the first and second steps of the reaction. Coordination of both required magnesium ions to the carboxylate of the substrate/product indicates a role for these cations in stabilization of the intermediate.     

The structure of a bacterial L-amino acid oxidase from Rhodococcus opacus gives new evidence for the hydride mechanism for dehydrogenation

l-Amino acid oxidase from Rhodococcus opacus (roLAAO) is classified as a member of the GR(2)-family of flavin-dependent oxidoreductases according to a highly conserved sequence motif for the cofactor binding. The monomer of the homodimeric enzyme consists of three well-defined domains: the FAD-binding domain corresponding to a general topology throughout the whole GR(2)-family; a substrate-binding domain with almost the same topology as the snake venom LAAO and a helical domain exclusively responsible for the unusual dimerisation mode of the enzyme and not found in other members of the family so far. We describe here high-resolution structures of the binary complex of protein and cofactor as well as the ternary complexes of protein, cofactor and ligands. This structures in addition to the structural knowledge of snake venom LAAO and DAAO from yeast and pig kidney permit more insight into different steps in the reaction mechanism of this class of enzymes. There is strong evidence for hydride transfer as the mechanism of dehydrogenation. This mechanism appears to be uncommon in a sense that the chemical transformation can proceed efficiently without the involvement of amino acid functional groups. Most groups present at the active site are involved in substrate recognition, binding and fixation, i.e. they direct the trajectory of the interacting orbitals. In this mode of catalysis orbital steering/interactions are the predominant factors for the chemical step(s). A mirror-symmetrical relationship between the two substrate-binding sites of d and l-amino acid oxidases is observed which facilitates enantiomeric selectivity while preserving a common arrangement of the residues in the active site. These results are of general relevance for the mechanism of flavoproteins and lead to the proposal of a common dehydrogenation step in the mechanism for l and d-amino acid oxidases.     

The medical utility of genomics data in neuropsychiatry: mutational genetics versus association genetics

The long anticipated ‘genetic revolution’ in neuropsychiatry has yet to have an impact on the practice of clinical medicine. Excitement in the 1980s over major genetic breakthroughs in schizophrenia and manic depression, for example, has been replaced in the late 1990s by the sobering realization that most common neuropsychiatric disorders are multifactorial. Despite considerable effort and resources, no ‘causative’ genetic variation has been identified that plays a definitive major role in any common neuropsychiatric disorder.     

Linkage disequilibrium mapping of complex disease: fantasy or reality?

In the past year, data about the level and nature of linkage disequilibrium between alleles of tightly linked SNPs have started to become available. Furthermore, increasing evidence of allelic heterogeneity at the loci predisposing to complex disease has been observed, which has lead to initial attempts to develop methods of linkage disequilibrium detection allowing for this difficulty. It has also become more obvious that we will need to think carefully about the types of populations we need to analyze in an attempt to identify these elusive genes, and it is becoming clear that we need to carefully reevaluate the prognosis of the current paradigm with regard to its robustness to the types of problems that are likely to exist.     

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

Applications of DNA shuffling to pharmaceuticals and vaccines

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.     

Structure of -amino acid oxidase: new insights from an old enzyme

-amino acid oxidase is the prototype of flavin-dependent oxidases. The recent resolution of its 3D structure has provided an explanation for several of its properties and has led to a substantial revision of the mechanism of -amino acid dehydrogenation, with significant implications for the general uderstanding of flavin-dependent catalysis     

Inkjet dispensing technology: applications in drug discovery

The past year has seen significant advances in the reduction to practice of inkjet dispensing technology in drug discovery applications. Although much of the work in this area has been done by relatively few ‘early innovators’, broader acceptance of the feasibility of the use of inkjet dispensing is on the rise. Of the three main areas of drug discovery — genomics, high-throughput screening, and combinatorial chemistry — high-throughput screening has had the most applications to date. The burgeoning field of genomics has seen rapid incorporation of technologies that enable miniaturization of gene expression experiments. Inkjet dispensing has a clear role in this effort. Finally, as the miniaturization needs of combinatorial chemistry become more clear, inkjet dispensing technology will potentially play a role.     

Muscle proteins — their actions and interactions

Muscle contracts by the myosin cross-bridges ‘rowing’ the actin filaments past the myosin filaments. In the past year many structural details of this mechanism have become clear. Structural studies indicate distinct states for myosin S1 in the rigor, ATP or ‘down’ conformation and in the products complex (ADP·P_i) or ‘up’ state. Crystallographic studies substantiate this classification and yield details of the transformation. The isomerization ‘up’ to ‘down’ is the power stroke of muscle. This consists in the main of large changes of angle of the ‘lever arm’ (at the distal part of the myosin head) which can account for an 11 nm power stroke.     

The chemical-biological interface: developments in automated and miniaturised screening technology

The rapidly changing developments in genomics and combinatorial chemistry, generating new drug targets and large numbers of compounds, have caused a revolution in high-throughput screening technologies. Key to this revolution has been the introduction of robotics and automation, together with new biological assay technologies (e.g., homogeneous time resolved fluorescence). With ever increasing workloads, together with economic and logistical constraints, miniaturisation is rapidly becoming essential for the future of high-throughput screening and combinatorial chemistry. This is evident from the introduction of high-density microtitre plates, small volume liquid handling robots and associated detection technology.     

The metaphase-to-anaphase transition: avoiding a mid-life crisis

The metaphase-to-anaphase transition is a highly regulated process, which is governed by the activity of the anaphase-promoting complex (APC). The APC promotes the degradation of several proteins, including mitotic cyclins and newly identified anaphase inhibitors. Several discoveries made this year shed invaluable light on the regulation of APC activation and its substrate specificity.     

Recognition of neutral species with synthetic receptors

The design of synthetic receptors for neutral molecules is an area of intense current interest. The area has grown from early work on cyclodextrin or single crown ether complexation to encompass a wide array of receptor shapes and structures. Furthermore, the range of substrate selectivities has increased from simple aromatic or metal ion substrates to include key biological components such as peptides and carbohydrates. Recent advances have included the application of split bead combinatorial methods for the identification of receptors for oligopeptides, the design of self-assembling spherical receptors, the use of multiple hydrogen bonding groups for carboxylic acid and carbohydrate recognition and the achievement of impressive catalytic effects with synthetic receptors.     

Ion channels in the immune system as targets for immunosuppression

The discovery of a diverse and unique subset of ion channels in T lymphocytes has led to a rapidly growing body of knowledge about their functional roles in the immune system. Potent and specific blockers have provided molecules tools to probe channel structure—function relations and to elucidate the involvement of K⁺, Ca²⁺, and Cl⁻ channels in T-cell activation and cell volume regulation. Recent advances in analyzing Kv1.3 channel structure—function relationships have defined binding sites for channel blockers, which have now been shown to be effective in suppressing T-cell function in vivo. Ion channels may provide excellent pharmaceutical targets for modulating immune system function.     

Vicious circles: a mechanism for yeast aging

The past year has confirmed the great potential of the yeast Saccharomyces cerevisiae as a model to study aging. Ground breaking papers have revealed similarities between aging in yeast and in mammals, and have identified genetic instability of the ribosomal DNA array as the first known cause of aging in yeast cells.     

Mitochondrial creatine kinase - a square protein

The recently determined structure of octameric mitochondrial creatine kinase has provided new insights into the functioning of this enzyme and its role in channelling energy from the mitochondria to the cytoplasm. Creatine kinase, a member of the family of guanidino kinases, is structurally similar to glutamine synthetase, suggesting a possible evolutionary link between both protein families     

Structure and dynamics of green fluorescent protein

Many marine organisms are luminescent. The proteins that produce the light include a primary light producer (aequorin or luciferase) and often a secondary photoprotein that red shifts the light for better penetration in the ocean. Green fluorescent protein is one such secondary protein. It is remarkable in that it autocatalyzes the formation of its own fluorophore and thus can be expressed in variety of organisms in its fluorescent form. The recent determination of its 3D structure and other physical characterizations are revealing its molecular mechanism of action