Current Chemical Biology - Volume 3, Issue 2, 2009

Oncogenic inhibition of the p53 tumour suppressor is a key feature of human cancer. However, relatively few oncogenic drug targets which inhibit p53 have been identified using a variety of model systems. As a prototype, a clinical proteomics screen was initiated in preneoplastic disease where selection pressures were placed on p53 gene mutation to identify clinically relevant and novel p53 inhibitors. This screen identified a protein named Anterior Gradient-2 (AGR2) as a novel type of p53 inhibitor. As reactivation of p53 holds promise as a therapeutic strategy for cancer treatment, AGR2 still requires validation as an anti-cancer drug target. Many signalling proteins function by binding to linear domains and combinatorial peptide libraries facilitate lead identification for validating a potential drug target. The AGR2 protein localized in the endoplasmic reticulum and nuclear compartments and this distribution was mediated by a Cterminal KTEL sequence. The transfection of AGR2 into cells stimulated p53 protein accumulation in the cytosol forming a novel assay for measuring AGR2-mediated inhibition of p53. Optimized AGR2-binding penta-peptide aptamers were minimized using substitution mutagenesis. Transfection of EGFP-peptide-aptamers containing the core TxIYY pentapeptide sequence stabilized p53 and increased its nuclear localization. The fusion of the TxIYY penta-peptide to the cell membrane permeable antennapedia peptide also resulted in a similar nuclear relocalization of p53 protein. These data indicate that the AGR2 pathway can be targeted with peptide-aptamers resulting in p53 stabilization and highlight the utility of linking clinical proteomics to combinatorial peptide chemistry to rapidly discover and validate uncharacterized proteins as potential anti-cancer drug targets.

S100A1 is a member of the S100 family of calcium-binding proteins. As with most S100 proteins, S100A1 undergoes a large conformational change upon binding calcium as necessary to interact with numerous protein targets. Targets of S100A1 include proteins involved in calcium signaling (ryanodine receptors 1 & 2, Serca2a, phopholamban), neurotransmitter release (synapsins I & II), cytoskeletal and filament associated proteins (CapZ, microtubules, intermediate filaments, tau, mocrofilaments, desmin, tubulin, F-actin, titin, and the glial fibrillary acidic protein GFAP), transcription factors and their regulators (e.g. myoD, p53), enzymes (e.g. aldolase, phosphoglucomutase, malate dehydrogenase, glycogen phosphorylase, photoreceptor guanyl cyclases, adenylate cyclases, glyceraldehydes-3-phosphate dehydrogenase, twitchin kinase, Ndr kinase, and F1 ATP synthase), and other Ca2+-activated proteins (annexins V & VI, S100B, S100A4, S100P, and other S100 proteins). There is also a growing interest in developing inhibitors of S100A1 since they may be beneficial for treating a variety of human diseases including neurological diseases, diabetes mellitus, heart failure, and several types of cancer. The absence of significant phenotypes in S100A1 knockout mice provides some early indication that an S100A1 antagonist could have minimal side effects in normal tissues. However, development of S100A1-mediated therapies is complicated by S100A1's unusual ability to function as both an intracellular signaling molecule and as a secreted protein. Additionally, many S100A1 protein targets have only recently been identified, and so fully characterizing both these S100A1-target complexes and their resulting functions is a necessary prerequisite.

Protein-protein interactions (PPI) play an important role in cellular signalling, the most common of which is the PDZ domain. This 90-residue domain occurs 257 times in 142 human proteins. Although these domains have well-defined binding sites which recognises short C-terminal peptide ligands (PLs), the high specificity of PDZ binding makes them an intriguing system to study. Abnormal PPI are of particular importance as they often contribute to the development of diseases. PDZ domains have been reported to be implicated in diseases such as cancer, cystic fibrosis, schizophrenia, Alzheimer's, Parkinson, avian influenza, pain, and stroke. In this review, we present the structure of PDZ domains, highlighting the high specificity of their binding to endogenous PLs, leading to their classification. We also consider their involvement in disease pathways and expand on current efforts to modulate their interaction using modified peptides and small molecules as selective PDZ inhibitors.

11β-Hydroxysteroid dehydrogenase (11β-HSD1) is involved in glucocorticoid activation in a tissue-specific manner. In tissues and intact cells, it functions primarily as a reductase by converting inactive cortisone to cortisol. Since glucocorticoid action is implicated in the development of the metabolic syndrome, inhibition of 11β-HSD1 has been proposed as a therapeutic strategy to treat type 2 diabetes. 11β-HSD1 is a member of the short chain dehydrogenase/reductase (SDR) superfamily with the “Y183SASK187” sequence and Ser170 at the active site that matches the catalytic domain signature motif of “YXXXK” in SDRs. The recent success in the determination of various 11β-HSD1 3-dimentional structures has facilitated a higher level understanding of the interactions among the enzyme, the cofactor and the substrate as well as substrate site inhibitors. In particular, the structural analysis of 11β-HSD1 in complex with a non-selective inhibitor carbenoxolone (CBX) has advanced our knowledge of inhibitor-enzyme interactions. In addition to the catalytic triad, residues close to the steroid binding site and surrounding areas have been identified as important in stabilizing the substrate. These findings have accelerated medicinal chemistry efforts to discover selective 11β-HSD1 inhibitors in the pharmaceutical industry. A large number of chemical classes have been identified as potent and selective 11β-HSD1 inhibitors. Some common features in enzyme-inhibitor interactions were observed with structurally distinct compounds. Chemical class-specific interactions were discovered as well, which explain the structural diversity of selective 11β-HSD1 inhibitors. In this article, we review the structural characteristics of 11β-HSD1 enzymes in several species. By comparing active sites, substrate binding and subunit interactions, we have identified structural features and residues in the active site important for inhibitor binding. Further, through analysis of the major interactions between the active site residues and inhibitors of different chemical classes, we have determined compound class-specific structural requirements for optimal enzyme- inhibitor interaction.

The elucidation of the manner in which proteins interact with other proteins or nucleic acids can lead to new drug and therapeutic leads in biological systems like signal transduction, protein targeting, and cell cycle control. Moreover, it aids in the design of experiments like mutagenesis and competition for binding sites and computer models to shed light on multi-protein interaction networks. Protein arrays are instrumental to analyze the interactions within complex protein networks in order to address these key questions in cellular biology. In this review, we summarize recent achievements in the field of solid-supported peptide arrays generated by the SPOT method, which allows for fast parallel manual or automated Fmoc-synthesis of arrays of overlapping oligopeptides on cellulose membranes. These arrays represent a powerful tool for the investigation/mapping of antibody epitopes, protein-protein interaction sites, protein-DNA interactions, and most recently protein-RNA interactions. We explain the method, provide an overview over the applications of the technique, and present recent experimental data, like the identification of a peptide sequence, that interferes with packaging of HIV-1 genomes and virus replication. Moreover, we summarize recent developments in the array formats used and discuss future perspectives of the method.

The uncoupling proteins (UCPs) are mitochondrial transporters that, according to their family name, should lower the efficiency of the oxidative phosphorylation. The uncoupling protein from brown adipose tissue UCP1 is the best characterized member of the family. It is a proton carrier, activated by low fatty acid concentrations, that has a thermogenic function. The physiological role of the uncoupling protein UCP2 is probably related to control of the production of reactive oxygen species since it is upregulated in situations of oxidative stress. This uncoupling activity would explain a higher respiratory activity to minimize the generation of superoxide or the modulation of the ATP levels in β-cells to control insulin secretion. The activity of UCP3 appears linked to lipid metabolism in muscle. The role could also be as a defence against oxidative stress although it has also been hypothesized to prevent fatty acid accumulation inside mitochondria. Fatty acids, when present at high concentrations, are known to interact with UCP2 and UCP3 but also with other mitochondrial carriers and therefore the specificity of those interactions is under debate. In this article we review our current understanding of the mechanism of transport, regulation and function of the mammalian uncoupling proteins.

Bone, a calcified tissue composed of 60% inorganic component (hydroxyapatite), 10% water and 30% organic component (proteins), has three functions: providing mechanical support for locomotion, protecting vital organs, and regulating mineral homeostasis. A lifelong execution of these functions depends on a healthy skeleton, which is maintained by constant bone remodeling in which old bone is removed by the bone-resorbing cell, osteoclasts, and then0 replaced by new bone formed by the bone-forming cell, osteoblasts. This remodeling process requires a physical interaction of bone with these bone cells. Moreover, numerous cancers including breast and prostate have a high tendency to metastasize to bone, which is in part attributable to the capacity of the tumor cells to attach to bone. The intensive investigation in the past two decades has led to the notion that the cell-bone interaction involves integrins on cell surface and bone matrix proteins. However, the biochemical composition of bone and emerging evidence are inconsistent with this belief. In this review, I will discuss the current understanding of the molecular mechanism underlying the cell-bone interaction. I will also highlight the facts and new findings supporting that the inorganic, rather than the organic, component of bone is likely responsible for cellular attachment.

Protection against free radical-initiated oxidative damage has long been recognized as the most important function of vitamin E. However, the mechanism by which vitamin E exerts its antioxidant function in vivo has yet to be delineated. Available information suggests that vitamin E may exert its antioxidant function at the tissue level by mediating the levels or generation of mitochondrial superoxide. Mitochondrion is the major source of superoxide, and high concentration of vitamin E is found in the inner membrane. Superoxide is a key precursor for such biologically important reactive oxygen species as hydrogen peroxide and peroxynitrite, and is capable of releasing iron from its protein complexes. The labile or free form of iron, in turn, can catalyze the formation of hydroxyl radicals from hydrogen peroxide. Both hydroxyl radicals and peroxynitrite have potential to initiate oxidative damage to essential biomembranes, proteins and DNA. Recently dietary vitamin E has been shown to reduce the levels/generation of mitochondrial superoxide, and to attenuate the tissue levels of labile iron. By reducing available superoxide, dietary vitamin E may decrease the generation of hydroxyl radicals and peroxynitrite, and thus attenuate oxidative damage. Also, by altering the levels/generation of reactive oxygen species, vitamin E may modulate the activation and/or expression of important redox-sensitive biological modifiers that mediate vital cellular events.

Chitin and its deacetylated derivative chitosan are natural polymers composed of randomly distributed β-(1-4)- linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitin is insoluble in aqueous media while chitosan is soluble in acidic conditions due to the free protonable amino groups present in the D-glucosamine units. Due to their natural origin, both chitin and chitosan can not be defined as a unique chemical structure but as a family of polymers which present a high variability in their chemical and physical properties. This variability is related not only to the origin of the samples but also to their method of preparation. Chitin and chitosan are used in fields as different as food, biomedicine and agriculture, among others. The success of chitin and chitosan in each of these specific applications is directly related to deep research into their physicochemical properties. In recent years, several reviews covering different aspects of the applications of chitin and chitosan have been published. However, these reviews have not taken into account the key role of the physicochemical properties of chitin and chitosan in their possible applications. The aim of this review is to highlight the relationship between the physicochemical properties of the polymers and their behaviour. A functional characterization of chitin and chitosan regarding some biological properties and some specific applications (drug delivery, tissue engineering, functional food, food preservative, biocatalyst immobilization, wastewater treatment, molecular imprinting and metal nanocomposites) is presented. The molecular mechanism of the biological properties such as biocompatibility, mucoadhesion, permeation enhancing effect, anticholesterolemic, and antimicrobial has been updated.

Mushroom development is important for both basic scientific research and for practical mushroom production. In previous screening studies of natural compounds that are effective for fruiting body development of oyster mushrooms (Pleurotus ostreatus) on agar medium, we identified a triterpenoid saponin that exerted a hormone-like effect. Not only did the saponin stimulate mushroom development, but P. ostreatus also changed its morphology as the concentration of saponin in the medium increased. We have synthesized a betulin glycoside by linking a sugar moiety to a triterpenoid betulin derived from outer bark of Betulla platyphylla. The sugar chain was further lengthened by transglycosylation using cyclodextrin glycosyltransferase (CGTase) of Bacillus macerans. Neither betulin nor the triterpenoid itself stimulated fruiting body development, but the activity was generated as a sugar moiety was introduced onto betulin. Fruiting activity correlated with the sugar length, indicating that the presence of the sugar moiety was indispensable for fruiting activity. We proposed that an as yet unknown hydrophobic compound with an attached sugar chain triggers fruiting in P. ostreatus. We synthesized a 3-O-alkyl-D-glucose (AG) and determined that AG with an eight-carbon alkyl chain effectively induced fruiting bodies for P. ostreatus. Thus, a sugar-containing amphipathic compound can stimulate fruiting body development.