Current Chemical Biology - Volume 1, Issue 2, 2007

Translational initiation sites in eukaryotic mRNAs are reached via a scanning mechanism which predicts that translation should start at the AUG codon nearest the 5' end of the mRNA. During the translational initiation in eukaryotic cell, the small (40S) ribosomal subunit binds to the 5' end of the mRNA. The 40S ribosomal subunit, carrying MettRNAi* eIF2*GTP complex and other factors, then migrates through the 5' UTR until it encounters the first AUG codon, which is recognized by base pairing with the anticodon in Met-tRNAi. Recently interest has focused on mechanisms that occasionally allow escape from this first-AUG rule. Approximately 10% of eukaryotic mRNAs skip this first AUG if it is in an unfavorable sequence context, and translation starts from the second or any downstream AUG codon. Furthermore, there is an increasing number of evidence to show that scanning translational initiation may start from a non-AUG codon that precedes or follow the first AUG codon in the mRNA. The initiation from non-AUG codons was discovered among viral genes, but newly reported examples include eukaryotic genes like those for some proto-oncogenes, transcription factor kinases and growth factors. Previously it was shown that regardless of the initiation codon used, methionine appears to be the initiating residue in all examples that have been investigated in eukaryotes. Recently few published results and our experiments showed that leucine can be translated as an initiator amino acid using CUG codon. Genome analysis generally considers the first AUG triplet in the open reading frame as the translation initiation codon. However the recent results indicate that non-AUG translation initiation may be operable more often than has been anticipated. This may have a great impact on the analysis of gene function based on genome information.

Boron neutron capture therapy (BNCT) is a promising treatment for malignant brain tumors as well as other cancers. Current research centers on both the design and synthesis of high boron containing compounds as BNCT agents, and the search for more effective delivery vehicles. This review discusses recent work on the development of nanomaterial based BNCT agents.

Xeroderma pigmentosum group A (XPA) and replication protein A (RPA) are two essential proteins for nucleotide excision repair (NER), a DNA repair pathway that removes a large variety of bulky DNA lesions in cells. In addition to its role in NER, RPA also is required for almost all other cellular DNA metabolic pathways, such as DNA replication, recombination, and other repair pathways. Although both proteins have been extensively studied for more than a decade, efforts have been focused mostly on their roles in DNA damage recognition in NER and/or single-stranded DNA binding. Recent advance in understanding the cellular functions of XPA and RPA reveals novel activities of the proteins in DNA damage responses. Briefly, XPA was found to be recruited to a DNA damage site for repair after TFIIH binds. The protein is also able to recognize specific structures of undamaged DNA. In addition, a nuclear import of XPA occurs upon DNA damage and in an ataxia-telangiectasia mutated and Rad3-related (ATR)-dependent manner. Furthermore, XPA undergoes phosphorylation by ATR checkpoint, promoting cell survival in response to UV irradiation. For RPA, the protein was found to play a role in activation of DNA damage checkpoint apparatuses ATR and the Rad9/Rad1/Hus1 complex. RPA also undergoes hyperphosphorylation upon DNA damage, which induces structural changes of the protein. Finally the hyperphosphorylation appears to be involved in modulating the activities of RPA and, thus, the cellular processes in which it participates. Here the molecular mechanisms by which XPA and RPA function in DNA damage responses are discussed in light of our recent understandings.

Cells have evolved specific intracellular compartments that permit local concentration of macromolecules. These macromolecules are transported from one part of the cell to another and eventually released into the extracellular space to participate in cell-to-cell communication. Neurons and neuroendocrine cells secrete neurotransmitters and hormones by exocytosis, a highly regulated process in which secretory vesicles fuse with the plasma membrane to release their contents in response to a calcium trigger. To date, many proteins that catalyze the formation, targeting and fusion of secretory vesicles have been identified. However, the lipid composition of vesicles and their target membrane is also critical and lipid modifications may be required at several stages of the exocytotic pathway. In this review, we will discuss the latest results suggesting important functions for cholesterol, phosphatidic acid (PA) and phosphatidylinositol 4,5- bisphosphate (PIP2) in the membrane merging process. We propose that exocytotic sites are determined by the local formation of lipid micro-domains, which are potentially important to allow structural and spatial organization of the exocytotic machinery. Among the lipid candidates, our results show that PA plays a decisive role in the late stages of exocytosis, most likely by changing the membrane curvature that may be required for membrane fusion to occur. The spatial and temporal coordination of the various players to form an efficient machinery for secretion now needs to be determined.

Resonance Energy Transfer (RET) plays an important role, both scientifically and commercially, in diagnostics and high throughput screening. For qualitative and quantitative analysis, RET systems are usually assembled through molecular recognition of biomolecules labeled with donor and acceptor luminophores. Lanthanide complexes, as well as quantum dot nanocrystals (QD), possess unique photophysical properties that make them especially suitable for applied RET systems in chemical biology. This review deals with the RET theory, and advantages are compared to conventional systems (using optical and other detection techniques). Different molecular recognition processes, as well as labeling techniques yielding biocompatibility are described. The photophysics of Ln complexes (e.g. millisecond luminescence decay times, line-shaped emission spectra, antenna effect of the ligand) and of QD (e.g. high extinction coefficients, size-tunable emission spectra, chemical stability) as well as their RET properties are described in detail. We give an overview of biochemical applications using lanthanide complexes and QD, e.g. immunoassays, DNA analysis and nanometer distance measurements (spectroscopic ruler) and some selected results are outlined. In particular, the recent scientific progress in biocompatible QD RET systems with the use of QD as energy donors as well as acceptors together with Ln complexes as donors is highlighted. The worldwide economic and scientific interests, as well as potentials for in vitro diagnostics (IVD) are addressed and the benefits regarding high throughput techniques, ultrahigh sensitivity, multiplexing measurements and miniaturization are discussed.

Carbohydrates in living cells and organisms are involved in various physiological and pathogenic processes through specific interactions with proteins. As a result, studies of carbohydrate-protein interactions not only provide valuable information for the understanding of biological phenomena but they also have the potential of leading to the development of novel carbohydrate-based pharmaceutical agents. The significance of these biomolecular interactions has stimulated exploitation for new technologies to rapidly analyze carbohydrate-mediated biological processes. In recent years, much effort has been made to develop carbohydrate microarrays which can be used for this purpose. The advancements made in this area have extended the scope of biological and biomedical research on carbohydrate-mediated molecular interactions. In this review, we describe progress that has been made in fabrication of carbohydrate microarrays, as well as in various applications of this technique to functional glycomics. In addition, recent progress of lectin microarrays for uses in glycoprofiling of glycoproteins, cells and pathogens is also described.

The completion of whole human genome sequencing encourages the development of a powerful gene delivery technology for elucidating structure, regulation and function of genes and proteins in addition to the emerging biomedical applications, such as industry-based productions of therapeutic proteins and ‘gene therapy’. Due to some major limitations of viral-mediated delivery, non-viral synthetic systems have become increasingly desirable. However, synthetic systems are notably inefficient compared to the viral ones in gene delivery and expression. We have recently developed the simplest, but highly efficient gene delivery device based on generated carbonate apatite nano-crystals having high affinity for DNA but fast dissolution kinetics for effective release of DNA during vesicular acidification and thus resulting in 5 to 100-fold higher transgene expression than the existing ones. Since like carbonate, Mg2+ incorporation into apatite could regulate particle size and solubility, we have also successfully designed nano-precipitates of Ca-Mg phosphate as much more efficient carriers of genetic materials than classical Ca phosphate precipitation method. Moreover, for cell-specific and more efficient transgene delivery, we successfully assembled a desirable cell-recognizable protein in the flexible manner and a highly hydrophilic protein onto the DNA/crystal surfaces, thereby creating dual surface properties, one facilitating cell-specific delivery and the other blocking non-specific interactions. Thus, considering the efficiency, celltargetability, biodegradability and simplicity, this newly developed gene delivery technology would emerge as a superior tool over the other existing ones for both basic research laboratories and clinical medicine and additionally, would enable to develop a new era for inorganic crystal-based therapeutic delivery.

Ras proteins belong to the superfamily of small GTP-binding proteins which have the ability to recognize and bind to several sets of effector proteins, thereby communicating signals into different pathways. Although the Ras proteins have almost identical amino acid residues in the effector binding region, and the Ras binding domains have a similar ubiquitin-like topology, the affinity constants span three orders of magnitude. Moreover, large differences in the individual enthalpic and entropic contributions are observed. Another important feature of Ras-effector interactions is the charge complementarity found between the proteins in the complex. As a result, the association rate constant is very high and contributes significantly to the affinity of the complex. Differences in binding affinities are mainly due to changes in the association rate constants with the dissociation rate constants being at a similar range. Here, we discuss the importance of understanding protein complex formation in signal transduction pathways on a molecular level, both, thermodynamically and kinetically. Although the focus is mainly on Ras-effector interactions, comparisons of different binding modes, thermodynamics and electrostatics of various effectors in complex with members of the Rho, Rab, Arf, and Ran families will also be considered. This information could be important to understand the specificity in different pathways and for a rational design of compounds which block specific pathways.

The Wnt signaling pathway regulates embryonic development, cell proliferation and cellular morphology. Deregulation and inappropriate activation of the pathway are associated with several diseases including cancer as well as bone and cartilage diseases such as arthritis. The high medical relevance of the Wnt pathway turns it into a tempting target for drug intervention. In this review we will summarize the major functions of the classical Wnt pathway with focus on its role in tumorigenesis. The proto-oncoproteins and tumor suppressor proteins of the Wnt pathway, which might serve as targets for therapeutic intervention, will be introduced. Despite many difficulties, a few promising achievements have been made over the last few years. Remarkable technical developments allowed the screening for compounds, which interfere with components and the activity of the Wnt pathway. We will describe several small synthetic compounds with inhibitory effects on the Wnt pathway and their effectiveness in biochemical and cell biological assays will be summarized.