Nucleotide analogues as probes for DNA polymerases

Transmission of the genetic information from the parental DNA strand to the offspring is crucial for the survival of any living species. In nature, all DNA synthesis in DNA replication, recombination and repair is catalyzed by DNA polymerases and depends on their ability to select the canonical nucleobase pair from a pool of structurally similar building blocks. Recently, a wealth of valuable new insights into DNA polymerase mechanisms have been gained through application of carefully designed synthetic nucleotides and oligonucleotides in functional enzyme studies. The applied analogues exhibit features that differ in certain aspects from their natural counterparts and, thus, allow investigation of the involvement and efficacy of a chosen particular aspect on the entire complex enzyme mechanism. This review will focus on a depiction of the efforts that have been undertaken towards the development of nucleotide analogues with carefully altered properties. The different approaches will be discussed in the context of the motivation and the problem under investigation.Received 16 March 2005; received after revision 5 May 2005; accepted 8 June 2005     

Y-family DNA polymerases in mammalian cells

Eukaryotic genomes are replicated with high fidelity to assure the faithful transmission of genetic information from one generation to the next. The accuracy of replication relies heavily on the ability of replicative DNA polymerases to efficiently select correct nucleotides for the polymerization reaction and, using their intrinsic exonuclease activities, to excise mistakenly incorporated nucleotides. Cells also possess a variety of specialized DNA polymerases that, by a process called translesion DNA synthesis (TLS), help overcome replication blocks when unrepaired DNA lesions stall the replication machinery. This review considers the properties of the Y-family (a subset of specialized DNA polymerases) and their roles in modulating spontaneous and genotoxic-induced mutations in mammals. We also review recent insights into the molecular mechanisms that regulate PCNA monoubiquitination and DNA polymerase switching during TLS and discuss the potential of using Y-family DNA polymerases as novel targets for cancer prevention and therapy.     

β-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome

Staphylococci have two mechanisms for resistance to β-lactam antibiotics. One is the production of β-lactamases, enzymes that hydrolytically destroy β-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by β-lactam antibiotics. Strains of S. aureus exhibiting either β-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism. Received 2 April 2005; received after revision 15 July 2005; accepted 25 July 2005     

Kallmann’s syndrome, a neuronal migration defect

Infertility and inability to smell are the phenotypical features of Kallmann’s syndrome (KS), a genetic disease which affects 1 in 10,000 males and 1 in 50,000 females, the majority of the cases being sporadic. The molecular pathogenesis of KS is complex but mainly referable to the impairment of olfactory axon development and of the migration of gonadotropin-releasing hormone (GnRH) neurons. Only two different genes have been identified so far as responsible for the disease: KAL1 and KAL2, encoding anosmin-1 and fibroblast growth factor receptor 1 (FGFR1), respectively. In this review we focus our attention on insights evoked by recent studies, which propose a new direct role for anosmin-1 in the migration GnRH neurons, and a fascinating hypothesis of interactions between anosmin-1 and FGFR1 systems. Received 23 December 2005; received after revision 31 May 2006; accepted 6 July 2006     

Inhibition by transmembrane peptides of chimeric insulin receptors

Receptor tyrosine kinases play essential roles in cell proliferation and differentiation. We have recently shown that peptides corresponding to the transmembrane domains of the epidermal growth factor (EGF) and ErbB2 receptors inhibit their corresponding receptor activation in cancer cell lines. We extend this observation to cells transfected with chimeric insulin receptors where the transmembrane domain has been replaced by that of the EGF receptor or a mutated Erb2 domain. Peptides corresponding to the transmembrane domains of the EGF receptor and ErbB2 are able to inhibit specifically the autophosphorylation of insulin receptors with the corresponding domain. This inhibitory effect is correlated with the propensity of the different transmembrane domains to self-associate in a genetic reporter assay. Thus, our data strengthen the notion that transmembrane domains are involved in erbB receptor activation, and that these receptors can be modulated by inhibiting proteinprotein interactions within the membrane.Received 25 May 2005; received after revision 13 July 2005; accepted 22 July 2005     

Unveiling molecular mechanisms of bacterial surface proteins: <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> as a model organism for structural studies

Bacteria present a variety of molecules either on their surface or in a cell-free form. These molecules take part in numerous processes in the interactions with their host, with its tissues and other molecules. These molecules are essential to bacterial pathogenesis either during colonization or the spread/invasion stages, and most are virulence factors. This review is focused on such molecules using Streptococcus pneumoniae, a Gram-positive bacterium, as an example. Selected surface proteins are introduced, their structure described, and, whenever available, their mechanisms of function on an atomic level are explained. Such mechanisms for hyaluronate lyase, pneumococcal surface protein A, pneumolysin, histidine-triad and fibronectin-binding proteins are discussed. Elucidation of molecular mechanisms of virulence factors is essential for the understanding of bacteria and their functional properties. Structural biology appears pivotal for these studies, as structural and mechanistic insights facilitate rational approach to the development of new treatments. Received 12 March 2007; received after revision 28 June 2007; accepted 18 July 2007     

Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment

The fatality of cancer predominantly results from the dissemination of primary tumor cells to distant sites and the subsequent formation of metastases. During tumor progression, some of the primary tumor cells as well as the tumor microenvironment undergo characteristic molecular changes, which are essential for the metastatic dissemination of tumor cells. In this review, we will discuss recent insights into pro-metastatic events occurring in tumor cells themselves and in the tumor stroma. Tumor cell-intrinsic alterations include the loss of cell polarity and alterations in cell-cell and cell-matrix adhesion as well as deregulated receptor kinase signaling, which together support detachment, migration and invasion of tumor cells. On the other hand, the tumor stroma, including endothelial cells, fibroblasts and cells of the immune system, is engaged in an active molecular crosstalk within the tumor microenvironment. Subsequent activation of blood vessel and lymph vessel angiogenesis together with inflammatory and immune-suppressive responses further promotes cancer cell migration and invasion, as well as initiation of the metastatic process. Received 4 July 2005; received after revision 3 November 2005; accepted 14 November 2005     

Retinoblastoma family proteins: insights gained through genetic manipulation of mice

The retinoblastoma (Rb) gene was identified as the first tumor suppressor gene two decades ago. Since this initial discovery, it has become clear that deregulated Rb function constitutes a hallmark of human malignancies. Rb is a well-established regulator of the cell cycle. Rb has also been implicated in playing a role in a wide variety of cellular processes including DNA repair, cellular senescence, cell fate determination and apoptosis. Animals lacking Rb and/or its family members p107 and p130 have led scientists to uncover new and exciting roles for this protein family in development as well as tumor suppression. The ability to ablate Rb in a temporal and cell-type-specific manner has offered further, often unexpected, insights into Rb function. This review summarizes the phenotypic consequences of Rb family ablation in mice, and discusses how these findings contribute to the increasingly complex picture of Rb family function in development and tumor suppression. Received 11 October 2005; received after revision 16 November 2005; accepted 28 November 2005     

Human selenoproteins at a glance

The public perception of selenium has changed significantly over the last decades. Originally mainly known for its high toxicity, it was later recognized as an essential trace element and is now (despite its narrow therapeutic window) almost being marketed as a lifestyle drug. Indeed, some clinical and preclinical studies suggest that selenium supplementation may be beneficial in a large number of clinical conditions. However, its mode of action is unresolved in most of these cases. Selenocysteine – identified as the 21^st amino acid used in ribosome-mediated protein synthesis – is incorporated in at least 25 specific, genetically determined human selenoproteins, many of which have only recently been discovered. Restoration of normal selenoprotein levels may be – apart from direct supranutritional effects – one possible explanation for the effects of selenium supplements. In this review we provide a brief but up-to-date overview of what is currently known about these 25 acknowledged human selenoproteins and their synthesis. Received 30 March 2005; received after revision 4 July 2005; accepted 13 July 2005     

Structural and dynamical features contributing to thermostability in α-amylases

In recent years an increasing number of studies on thermophilic and hyperthermophilic proteins aiming to elucidate determinants of protein thermostability have yielded valuable insights about the relevant mechanisms. In particular, comparison of homologous enzymes with different thermostabilities (isolated from psychrophilic, mesophilic, thermophilic and hyperthermophilic organsims) offers a unique opportunity to determine the strategies of thermal adaptation. In this respect, the medium-sized amylolytic enzyme α-amylase is a well-established representative. Various studies on α-amylases with very different thermostabilities (melting temperature T_m = 40–110°C) report structural and dynamical features as well as thermodynamical properties which are supposed to play key roles in thermal adaptation. Here, results from selected homologous α-amylases are presented and discussed with respect to some new and recently proposed strategies to achieve thermostability.Received 28 February 2005; received after revision 29 April 2005; accepted 19 May 2005     

Identification of factor XIII-A as a marker of alternative macrophage activation

Factor XIII subunit A of blood coagulation (FXIII-A) is known to be synthesized but not secreted by the monocyte/macrophage cell line. On the basis of its intracellular localization and substrate profile, FXIII-A is thought to be involved in certain intracellular processes. Our present study was designed to monitor the changes in FXIII-A gene expression and protein production in long-term culture of human monocytes during their differentiation into macrophages in the presence of activating agents (interleukin-4, interferon-γ, Mycobacterium bovis BCG) inducing classical and alternative activation pathways. By using quantitative RT-PCR and fluorescent image analysis at the single-cell level we demonstrated that the expression of FXIII-A both at the mRNA as well as at the protein level is inversely regulated during the two activation programmes. Here we conclude that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophages indicates their classically activated state.Received 2 June 2005; received after revision 12 July 2005; accepted 22 July 2005     

Isoprenylated proteins

Isoprenoids are synthesized in all living organisms and are incorporated into diverse classes of end-products that participate in a multitude of cellular processes relating to cell growth, differentiation, cytoskeletal function and vesicle trafficking. In humans, the non-sterol isoprenoids, farnesyl pyrophosphate and geranylgeranyl-pyrophosphate, are synthesized via the mevalonate pathway and are covalently added to members of the small G protein superfamily. Isoprenylated proteins have key roles in membrane attachment and protein functionality, have been shown to have a central role in some cancers and are likely also to be involved in the pathogenesis and progression of atherosclerosis and Alzheimer disease. This review details current knowledge on the biosynthesis of isoprenoids, their incorporation into proteins by the process known as prenylation and the complex regulatory network that controls these proteins. An improved understanding of these processe is likely to lead to the development of novel therapies that will have important implications for human health and disease. Received 5 July 2005; received after revision 17 October 2005; accepted 22 October 2005     

Functional and physical communication between oncoproteins and tumor suppressors

The discovery of oncogenes (c-onc’s) and tumor suppressors (TS’s) has led to the concept that cancer arises from defects in each of these classes of genes or their products. More recently, it has been appreciated that c-onc and TS proteins often affect one another’s functions. Within this context, I review the two classical TS’s, p53 and the retinoblastoma protein, and the consequences of their inactivation. The various forms of genomic instability (GI) that underly the high mutation rates of transformed cells are then discussed. Particular emphasis is placed upon the concept that GI is not only an integral part of the transformed state but is a prerequisite. Increased oxidative DNA damage, and/or an inabiliy to repair it, can lead to GI. The review then discusses recent observations showing that loss of the TS protein peroxiredoxin 1 (prdx1) and increased expression of the c-onc protein c-Myc, each leads to increased oxidative DNA damage. The critical nature of the c-onc-TS interaction is underscored by that occurring between prdx1 and c-Myc, with the former protein regulating the production of DNA-damaging reactive oxygen species by the latter. The intimate association between these proteins and others serves as a paradigm for the exquisite balancing act that c-onc’s and TS’s must maintain in order to properly control normal DNA replication and cellular proliferation while simultaneously minimizing the acquisition of potentially neoplastic mutations. Received 10 May 2005; received after revision 3 July 2005; accepted 19 July 2005     

Molecular guardians for newborn proteins: ribosome-associated chaperones and their role in protein folding

A central dogma in biology is the conversion of genetic information into active proteins. The biosynthesis of proteins by ribosomes and the subsequent folding of newly made proteins represent the last crucial steps in this process. To guarantee the correct folding of newly made proteins, a complex chaperone network is required in all cells. In concert with ongoing protein biosynthesis, ribosome-associated factors can interact directly with emerging nascent polypeptides to protect them from degradation or aggregation, to promote folding into their native structure, or to otherwise contribute to their folding program. Eukaryotic cells possess two major ribosome-associated systems, an Hsp70/Hsp40-based chaperone system and the functionally enigmatic NAC complex, whereas prokaryotes employ the Trigger Factor chaperone. Recent structural insights into Trigger Factor reveal an intricate cradle-like structure that, together with the exit site of the ribosome, forms a protected environment for the folding of newly synthesized proteins. Received 29 June 2005; received after revision 4 August 2005; accepted 18 August 2005     

Human progeroid syndromes,aging and cancer: new genetic and epigenetic insights into old questions

Disorders in which individuals exhibit certain features of aging early in life are referred to as segmental progeroid syndromes. With the progress that has been made in understanding the etiologies of these conditions in the past decade, potential therapeutic options have begun to move from the realm of improbability to initial stages of testing. Among these syndromes, relevant advances have recently been made in Werner syndrome, one of several progeroid syndromes characterized by defective DNA helicases, and Hutchinson-Gilford progeria syndrome, which is characterized by aberrant processing of the nuclear envelope protein lamin A. Although best known for their causative roles in these illnesses, Werner protein and lamin A have also recently emerged as key players vulnerable to epigenetic changes that contribute to tumorigenesis and aging. These advances further demonstrate that understanding progeroid syndromes and introducing adequate treatments will not only prove beneficial to patients suffering from these dramatic diseases, but will also provide new mechanistic insights into cancer and normal aging processes. Received 28 July 2006; received after revision 5 September 2006; accepted 13 October 2006     

δ-Protocadherins: unique structures and functions

δ-Protocadherins constitute a group of cadherins characterized by several conserved motifs in their cytoplasmic domains. We present a phylogenetic analysis that further divides this group into δ1-protocadherins (comprising protocadherin-1, −7, −9 and −11 or -X/Y) and δ2-protocadherins (comprising protocadherin-8, −10, −17, −18 and −19). The δ-protocadherin genes, which are located on different chromosomes in man and mouse, have a similar gene structure. They are expressed as multiple splice forms, differing mostly in their cytoplasmic domains. Some δ-protocadherins were reported to mediate weak cell-cell adhesion in vitro and cell sorting in vivo. In addition, individual δ-protocadherins might play important roles in signaling pathways, as they bind to proteins such as TAF1/Set, protein phosphatase-1α and the Frizzled 7 receptor. The spatiotemporally restricted expression of δ-protocadherins in different tissues and species and the results of their functional analysis, mainly in Xenopus, suggest that they play multiple, tightly regulated roles in vertebrate development. Received 18 July 2005; received after revision 26 August 2005; accepted 2 September 2005     

Protein flexibility: its role in structure and mechanism revealed by molecular simulations

Computer simulations at the atomic level have arrived at a stage where they provide realistic modeling of flexibility in proteins (and the mobility of their associated solvent) that is important in understanding the nature of molecular motions. This can now be extended to the molecular and atomic motions that are associated with protein mechanisms. Moreover, the derived data agree reasonably accurately with experimental measurements of several kinetic and thermodynamic parameters. Fundamental insights emerge on the roles that this intrinsic flexibility plays in the thermodynamic characteristics of macromolecules in solution; these equip the investigator to probe the consequences of cognate interactions and ligand binding on entropy and enthalpy. Thus simulations can now provide a powerful tool for investigating protein mechanisms that complements the existing and the emerging experimental techniques. Received 29 May 2005; received after revision 23 August 2005; accepted 21 October 2005     

Molecular analysis of axonal target specificity and synapse formation

The development of neuronal connectivity requires the growth of axons to their target region and the formation of dendritic trees that extend into specific layers. Within the target region growth cones, the tips of extending axons are guided to finer target fields including specific subcellular compartments where they form synapses. In this article we highlight recent progress on molecular aspects of axonal subcellular target selection such as the axon initial segment or specific sublaminae of the vertebrate retina. We then discuss the very recent progress on the molecular analysis of synapse formation in the central nervous system, including the direction of differentiation into an inhibitory or excitatory synapse. Apparently, initial synaptic contacts are structurally and functionally modulated by neuronal activity, raising the question how neuronal activity can modify synaptic circuits. We therefore also focus on neural proteins that are up-regulated, secreted or converted by synaptic activity and, thus, might represent molecular candidates for experience-driven refinement or remodeling of synaptic connections. Received 5 July 2005; received after revision 19 August 2005; accepted 2 September 2005