Myosin I: From yeast to human

Myosin I is a non-filamentous, single-headed, actin-binding motor protein and is present in a wide range of species from yeast to man. The role of these class I myosins have been studied extensively in simple eukaryotes, showing their role in diverse processes such as actin cytoskeleton organization, cell motility, and endocytosis. Recently, studies in metazoans have begun to reveal more specialized functions of myosin I. It will be a major challenge in the future to examine the physiological functions of each class I myosin in different cell types of metazoans.     

The antioxidant function of Bcl-2 preserves cytoskeletal stability of cells with defective respiratory complex I

Human thyroid carcinoma XTC.UC1 cells harbor a homoplasmic frameshift mutation in the MT-ND1 subunit of respiratory complex I. When forced to use exclusively oxidative phosphorylation for energy production by inhibiting glycolysis, these cells triggered a caspase-independent cell death pathway, which was associated to a significant imbalance in glutathione homeostasis and a cleavage of the actin cytoskeleton. Overexpression of the anti-apoptotic Bcl-2 protein significantly increased the level of endogenous reduced glutathione, thus preventing its oxidation after the metabolic stress. Furthermore, Bcl-2 completely inhibited actin cleavage and increased cell adhesion, but was unable to improve cellular viability. Similar effects were obtained when XTC.UC1 cells were incubated with exogenous glutathione. We hence propose that Bcl-2 can safeguard cytoskeletal stability through an antioxidant function. Received 28 May 2008; received after revision 8 July 2008; accepted 29 July 2008     

A complex of Shc and Ran-GTPase localises to the cell nucleus

The three isoforms of the adaptor protein Shc play diverse roles in cell signalling. For example, the observation of p46 Shc in the nuclei of hepatocellular carcinoma cells suggests a function quite distinct from the better characterised cytoplasmic role. Ligands responsible for the transport of various Shc isoforms into organelles such as the nucleus have yet to be reported. To identify such ligands a far western approach was used to determine the p52 Shc interactome. The Ran-GTPase nuclear transport protein was identified and found to bind to p52 Shc in vitro with low micromolar affinity. Co-immunoprecipitation, pull down and fluorescence lifetime imaging microscopy experiments in stable cells confirmed cellular interaction and nuclear localisation. The nuclear transport factor protein NTF2, which functions in cohort with Ran, was shown to form a complex with both RAN and Shc, suggesting a mechanism for Shc entry into the nucleus as part of a tertiary complex. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 20 October 2008; received after revision 04 December 2008; accepted 15 December 2008     

Epigenetic mechanisms in mammals

DNA and histone methylation are linked and subjected to mitotic inheritance in mammals. Yet how methylation is propagated and maintained between successive cell divisions is not fully understood. A series of enzyme families that can add methylation marks to cytosine nucleobases, and lysine and arginine amino acid residues has been discovered. Apart from methyltransferases, there are also histone modification enzymes and accessory proteins, which can facilitate and/or target epigenetic marks. Several lysine and arginine demethylases have been discovered recently, and the presence of an active DNA demethylase is speculated in mammalian cells. A mammalian methyl DNA binding protein MBD2 and de novo DNA methyltransferase DNMT3A and DNMT3B are shown experimentally to possess DNA demethylase activity. Thus, complex mammalian epigenetic mechanisms appear to be dynamic yet reversible along with a well-choreographed set of events that take place during mammalian development.     

The Dim protein family: from structure to splicing

The spliceosome is a dynamic macromolecular machine that catalyzes pre-mRNA splicing through a mechanism controlled by several accessory proteins, including the Dim proteins. The Dim protein family is composed of two classes, Dim1 and Dim2, which share a common thioredoxin-like fold. They were originally identified for their role in cell cycle progression and have been found to interact with Prp6, an essential component of the spliceosome, which forms the bridge of U4/U6.U5-tri-snRNP. In spite of their biological and structural similarities, Dim1 and Dim2 proteins differ in many aspects. Dim1 bears distinctive structural motifs responsible for its interaction with other spliceosome components. Dim2 forms homodimers and contains specific domains required for its interactions with partners. This originality suggests that although both proteins are involved in pre-mRNA splicing, they are likely to be involved in different biological pathways. In the present article we review the structure and function of the Dim proteins.     

Protein assemblies with palindromic structure motifs

Symmetric DNA sequence motifs allow the formation of palindromic protein/DNA complexes. Although symmetric protein sequence motifs are less common, recent structural discoveries have unraveled a few protein/protein complexes with palindromic symmetry. Remarkably, symmetric protein/protein complexes can be generated either by adjacent or remote sequence motifs, which may be repeated or inverted. This contribution reflects and comments on recent findings of palindromic protein/protein complexes. Received 14 May 2008; received after revision 21 June 2008; accepted 14 July 2008     

Src activation and translocation from focal adhesions to membrane ruffles contribute to formation of new adhesion sites

Cell migration requires the coordinated turnover of focal adhesions, a process that involves FAK phosphorylation. Since Src is the major kinase implicated in FAK phosphorylation, we focus here on the role of Src activation on adhesion remodelling. In astrocytoma cells, constitutively activated Src induces both FAK phosphorylation and adhesion rearrangement. To evaluate how Src controls these processes, we used a recently described Src reporter to monitor the dynamics of Src phosphorylation. Upon Src activation, focal adhesions started to disassemble while Src appeared highly expressed at newly formed membrane ruffles. Kinetic analysis of time-lapse movies showed that loss of phospho-Src at focal adhesions was time-correlated with the appearance of membrane ruffles containing phospho-Src. Moreover, FLIP analysis revealed a dynamic equilibrium of Src between focal adhesions and membrane ruffles. We conclude that upon phosphorylation, Src is directly translocated from focal adhesions to membrane ruffles, thereby promoting formation of new adhesion complexes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 21 July 2008; received after revision 10 October 2008; accepted 03 November 2008     

Bile Acids: Chemistry, Pathochemistry, Biology, Pathobiology, and Therapeutics

Bile acids and bile alcohols in the form of their conjugates are amphipathic end products of cholesterol metabolism with multiple physiological functions. The great variety of bile acids and bile alcohols that are present in vertebrates are tabulated. Bile salts have an enterohepatic circulation resulting from efficient vectorial transport of bile salts through the hepatocyte and the ileal enterocyte; such transport leads to the accumulation of a pool of bile salts that cycles between the liver and intestine. Bile salt anions promote lipid absorption, enhance tryptic cleavage of dietary proteins, and have antimicrobial effects. Bile salts are signaling molecules, activating nuclear receptors in the hepatocyte and ileal enterocyte, as well as an increasing number of G-protein coupled receptors. Bile acids are used therapeutically to correct deficiency states, to decrease the cholesterol saturation of bile, or to decrease the cytotoxicity of retained bile acids in cholestatic liver disease.     

Gene expression in spermiogenesis

Germ cells convey parental genes to the next generation, and only germ cells perform meiosis, which is a mechanism that preserves the parental genes. The fusion of the products of germ cell meiosis, the haploid sperm and egg, creates the next generation. Sperm are the haploid germ cells that contribute genes to the egg. In preparation for this, the haploid round spermatids produced by meiosis undergo drastic morphological changes to become sperm. During this process of spermiogenesis, the nuclear form of the haploid germ cell takes shape, the mitochondria are rearranged in a specific manner, the flagellum develops and the acrosome forms. Spermatogenesis is supported by precise and orderly regulation of gene expression during the changes in chromatin structure, when protamine replaces histone. In this report, we summarize the molecular mechanisms involved in spermiogenesis.Received 2 September 2004; received after revision 7 October 2004; accepted 7 October 2004     

hShroom1 links a membrane bound protein to the actin cytoskeleton

hShroom1 (hShrm1) is a member of the Apx/Shroom (Shrm) protein family and was identified from a yeast two-hybrid screen as a protein that interacts with the cytoplasmic domain of melanoma cell adhesion molecule (MCAM). The characteristic signature of the Shrm family is the presence of a unique domain, ASD2 (Apx/Shroom domain 2). mRNA analysis suggests that hShrm1 is expressed in brain, heart, skeletal muscle, colon, small intestine, kidney, placenta and lung tissue, as well a variety of melanoma and other cell lines. Co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments indicate that hShrm1 and MCAM interact in vivo and by immunofluorescence microscopy some co-localization of these proteins is observed. hShrm1 partly co-localises with β-actin and is found in the Triton X-100 insoluble fraction of melanoma cell extracts. We propose that hShrm1 is involved in linking MCAM to the cytoskeleton. D. E. Dye, S. Karlen: These authors contributed equally to this work. Received 09 October 2008; received after revision 23 November 2008; accepted 09 December 2008     

The Role of the Agouti-Related Protein in Energy Balance Regulation

The Agouti-Related Protein (AgRP) is a powerful orexigenic peptide that increases food intake when ubiquitously overexpressed or when administered centrally. AgRP-deficiency, on the other hand, leads to increased metabolic rate and a longer lifespan when mice consume a high fat diet. In humans, AgRP polymorphisms have been consistently associated with resistance to fatness in Blacks and Whites and resistance to the development of type-2 diabetes in African Blacks. Systemically administered AgRP accumulates in the liver, the adrenal gland and fat tissue while recent findings suggest that AgRP may also have inverse agonist effects, both centrally and peripherally. AgRP could thus modulate energy balance via different actions. Its absence or reduced functionality may offer a benefit both in terms of bringing about negative energy balance in obesigenic environments, as well as leading to an increased lifespan.     

A sperm GPI-anchored protein elicits sperm-cumulus cross-talk leading to the acrosome reaction

The acrosome reaction has long been thought to be induced by the zona pellucida. Here we report the identification and function of a novel human sperm glycosylphosphatidylinositol (GPI)-anchored membrane protein, NYD-SP8. The release of the protein during sperm-egg interaction and its binding to the cumulus, the first layer of egg investment, elicits cross-talk between the gametes and produces calcium dependant release of progesterone, which lead to the acrosome reaction. An in vivo mouse model of NYD-SP8 immunization is also established showing a reduced fertility rate. Thus, contrary to accepted dogma, our study demonstrates for the first time that, prior to reaching the zona pellucida, sperm may release a surface protein that acts on the cumulus cells leading to the acrosome reaction, which may be important for determining the outcome of fertilization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 11 August 2008; received after revision 18 December 2008; accepted 22 December 2008     

The many faces of semaphorins: from development to pathology

The semaphorin family is a large group of proteins controlling cell migration and axonal growth cone guidance. These proteins are bi-functional signals capable of growth promotion or growth inhibition. Initially described in the nervous system, the majority of studies related to semaphorins and semaphorin signalling are nowadays performed in model systems outside the nervous system. Here, we provide an exhaustive review of the many faces of semaphorins both during developmental, regulatory and pathological processes. Indeed, because of their crucial fundamental roles, the semaphorins and their receptors represent important targets for the development of drugs directed at a variety of diseases. Received 22 August 2008; received after revision 22 September 2008; accepted 24 September 2008 L. Roth, E. Koncina, S. Satkauskas: These authors contributed equally to this work.     

Dissection of the molecular mechanisms that control the nuclear accumulation of transport factors importin-α and CAS in stressed cells

The physiological state of eukaryotic cells controls nuclear trafficking of numerous cargos. For example, stress results in the inhibition of classical protein import, which is characterized by the redistribution of several transport factors. As such, importin-alpha and cellular apoptosis susceptibility protein (CAS) accumulate in nuclei of heat-shocked cells; however, the mechanisms underlying this relocation are not fully understood. We now show that heat upregulates the initial docking of importin-alpha at the nuclear envelope and stimulates the translocation of CAS into the nuclear interior. Moreover, heat exposure compromises the exit of importin-alpha from nuclei and drastically increases its retention in the nucleoplasm, whereas CAS nuclear exit and retention are less affected. Taken together, our results support the idea that heat shock regulates importin-alpha and CAS nuclear accumulation at several levels. The combination of different stress-induced changes leads to the nuclear concentration of both transport factors in heat-stressed cells.     

The chemical versatility of the β–α–β fold: Catalytic promiscuity and divergent evolution in the tautomerase superfamily

Tautomerase superfamily members have an amino-terminal proline and a β–α–β fold, and include 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), trans- and cis-3-chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF), which exhibits a phenylpyruvate tautomerase (PPT) activity. Pro-1 is a base (4-OT, CHMI, the PPT activity of MIF) or an acid (CaaD, cis-CaaD, MSAD). Components of the catalytic machinery have been identified and mechanistic hypotheses formulated. Characterization of new homologues shows that these mechanisms are incomplete. 4-OT, CaaD, cis-CaaD, and MSAD also have promiscuous activities with a hydratase activity in CaaD, cis-CaaD, and MSAD, PPT activity in CaaD and cis-CaaD, and CaaD and cis-CaaD activities in 4-OT. The shared promiscuous activities provide evidence for divergent evolution from a common ancestor, give hints about mechanistic relationships, and implicate catalytic promiscuity in the emergence of new enzymes. Received 22 May 2008; received after revision 20 June 2008; accepted 02 July 2008     

From endoderm to pancreas: a multistep journey

The formation of the vertebrate pancreas is a complex process that typifies the basic steps of embryonic development. It involves the establishment of competence, specification, signaling from neighboring tissues, morphogenesis, and the elaboration of tissue-specific genetic networks. A full analysis of this multistep process will help us to understand classic principles of embryonic development. Furthermore, this will provide the blueprint for experimental programming of pancreas formation from embryonic stem cells in the context of diabetes cell-therapy. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatogenesis, important gaps persist in our knowledge of early pancreas formation. This review will summarize the current understanding of the early mechanisms coming into play to pattern the "pre-pancreatic" region within the endoderm and, gradually, specify the pancreatic tissue.