Gene structure of the murine 2'-5'-oligoadenylate synthetase family

The 2'-5'-oligoadenylate synthetases (OASs) are members of a family of interferon-induced proteins playing an important role in the antiviral effect of interferons as well as being involved in apoptosis and control of cellular growth. Based on sequence data from the murine BAC clone (RP23-39M18), and a number of EST and IMAGE clones and the Celera Mouse database, we identified twelve Oas genes in the mouse genome, all localized to the chromosome 5F region. In contrast to the single OAS1 gene found in humans, we identified eight closely linked Oas1 genes in the murine genome, together with the genes of Oas2 and Oas3. Compared to the single OASL gene found in humans, two genes of OAS-like proteins, Oasl1 and Oasl2, were identified. All the putative genes seem to be transcribed. The exon/intron structures of the murine Oas genes were found to be identical to those of the human genes.     

MLL5 (KMT2E): structure,function, and clinical relevance

The mixed lineage leukemia (MLL) family of genes, also known as the lysine N-methyltransferase 2 (KMT2) family, are homologous to the evolutionarily conserved trithorax group that plays critical roles in the regulation of homeotic gene (HOX) expression and embryonic development. MLL5, assigned as KMT2E on the basis of its SET domain homology, was initially categorized under MLL (KMT2) family together with other six SET methyltransferase domain proteins (KMT2A–2D and 2F–2G). However, emerging evidence suggests that MLL5 is distinct from the other MLL (KMT2) family members, and the protein it encodes appears to lack intrinsic histone methyltransferase (HMT) activity towards histone substrates. MLL5 has been reported to play key roles in diverse biological processes, including cell cycle progression, genomic stability maintenance, adult hematopoiesis, and spermatogenesis. Recent studies of MLL5 variants and isoforms and putative MLL5 homologs in other species have enriched our understanding of the role of MLL5 in gene expression regulation, although the mechanism of action and physiological function of MLL5 remains poorly understood. In this review, we summarize recent research characterizing the structural features and biological roles of MLL5, and we highlight the potential implications of MLL5 dysfunction in human disease.     

Relationship between the structure of chloramphenicol and its action upon peptide synthetase

Zusammenfassung Zusammenhänge zwischen Struktur und Wirkung bei Chloramphenicol führen zu der Hypothese, dass das Antibiotikum die Peptidsynthetase der Ribosome dadurch hemmt, dass es den Peptidylpartner der Reaktion antagonisiert. Im Einklang damit steht, dass Chloramphenicol sich spezifisch an Ribosome bindet und selektiv die Elongationsphase der Proteinsynthese hemmt.     

Sterol carrier protein-2: structure reveals function

The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the hydrophobic nature of the previously ill-defined C-terminus suggested that these 20 amino acids may form a 'hydrophobic cap' which closes around the oleic acid upon binding. Thus, understanding the structural domains of the SCP-x/pro-SCP-2 gene and its respective posttranslationally processed proteins has provided new insights into their functions in intracellular targeting and metabolism of lipids.     

Protein structure and function

Posters

Protein structure and function     

RNA-splicing endonuclease structure and function

The RNA-splicing endonuclease is an evolutionarily conserved enzyme responsible for the excision of introns from nuclear transfer RNA (tRNA) and all archaeal RNAs. Since its first identification from yeast in the late 1970s, significant progress has been made toward understanding the biochemical mechanisms of this enzyme. Four families of the splicing endonucleases possessing the same active sites and overall architecture but with different subunit compositions have been identified. Two related consensus structures of the precursor RNA splice sites and the critical elements required for intron excision have been established. More recently, a glimpse was obtained of the structural mechanism by which the endonuclease recognizes the consensus RNA structures and cleaves at the splice sites. This review summarizes these findings and discusses their implications in the evolution of intron removal processes. Received 24 August 2007; received after revision 24 November 2007; accepted 27 November 2007     

Gene amplification and its effect on the structure and function of the oocyte nucleus in the whirligig beetleGyrinus natator (Gyrinidae,Coleoptera-Adephaga)

Zusammenfassung Autoradiographische Untersuchungen an den Oozytenkernen des TaumelkäfersGyrinus natator zeigen eine hohe RNA-Syntheseaktivität, kurz nachdem sich der extrachromosomale Chromatinkörper im Karyoplasma aufgelockert hat. Da die Oozytenchromosomen gleichzeitig in der Karyosphäre zusammengeballt sind, wird angenommen, dass die Transkriptionsaktivität an der extrachromosomalen DNA abläuft.

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This research was supported in part by funds from the Cytobiology Committe of the Polish Academy of Science.     

Unique gene structure and paralogy define the 7D-cadherin family

Cadherins are Ca²⁺-dependent transmembrane glycoproteins crucial for cell-cell adhesion in vertebrates and invertebrates. Classification of this superfamily due to their phylogenetic relationship is currently restricted to three major subfamilies: classical, desmosomal and protocadherins. Here we report evidence for a common phylogenetic origin of the kidney-specific Ksp- (Cdh16) and the intestine-specific LI-cadherin (Cdh17). Both genes consist of 18 exons and the positions of their exon-intron boundaries as well as their intron phases are perfectly conserved. We found an extensive paralogy of more than 40 megabases in mammals as well as teleost fish species encompassing the Ksp- and LI-cadherin genes. A comparable paralogy was not detected for other cadherin gene loci. These findings suggest that the Ksp- and LI-cadherin genes originated by chromosomal duplication early during vertebrate evolution and support our assumption that both proteins are paralogues within a separate cadherin family that we have termed 7D-cadherins. Received 16 January 2006; received after revision 18 April 2006; accepted 11 May 2006     

The CorA family: Structure and function revisited

The CorA family is a group of ion transporters that mediate transport of divalent metal ions across biological membranes. Metal ions are essential elements in most cellular processes and hence the concentrations of ions in cells and organelles must be kept at appropriate levels. Impairment of these systems is implied in a number of pathological conditions. CorA proteins are abundant among the prokaryotic organisms but homologues are present in both human and yeast. The activity of CorA proteins has generally been associated with the transport of magnesium ions but the members of the CorA family can also transport other ions such as cobalt and nickel. The structure of the CorA from Thermotoga maritima, which also was the first structure of a divalent cation transporter determined, has opened the possibilities for understanding the mechanisms behind the ion transport and also corrected a number of assumptions that have been made in the past.     

Intragenic complementation and the structure and function of argininosuccinate lyase

Argininosuccinate lyase (ASL) catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate, a reaction important for the detoxification of ammonia via the urea cycle and for arginine biosynthesis. ASL belongs to a superfamily of structurally related enzymes, all of which function as tetramers and catalyze similar reactions in which fumarate is one of the products. Genetic defects in the ASL gene result in the autosomal recessive disorder argininosuccinic aciduria. This disorder has considerable clinical and genetic heterogeneity and also exhibits extensive intragenic complementation. Intragenic complementation is a phenomenon that occurs when a multimeric protein is formed from subunits produced by different mutant alleles of a gene. The resulting hybrid protein exhibits greater enzymatic activity than is found in either of the homomeric mutant proteins. This review describes the structure and function of ASL and its homologue delta crystallin, the genetic defects associated with argininosuccinic aciduria and current theories regarding complementation in this protein.     

Ca2+ release-activated Ca2+ (CRAC) current, structure, and function

Store-operated Ca²⁺ entry describes the phenomenon that connects a depletion of internal Ca²⁺ stores to an activation of plasma membrane-located Ca²⁺ selective ion channels. Tremendous progress towards the underlying molecular mechanism came with the discovery of the two respective limiting components, STIM and Orai. STIM1 represents the ER-located Ca²⁺ sensor and transmits the signal of store depletion to the plasma membrane. Here it couples to and activates Orai, the highly Ca²⁺-selective pore-forming subunit of Ca²⁺ release-activated Ca²⁺ channels. In this review, we focus on the molecular steps that these two proteins undergo from store-depletion to their coupling, the activation, and regulation of Ca²⁺ currents.     

Insulin receptors: structure and function

The recent characterization of the human insulin receptor structure and its intrinsic tyrosine kinase activity represent major advances in our understanding of the mechanism of insulin action. It is reasonable to think that the insulin-induced autophosphorylation and activation of its receptor kinase represent an important event in the action of insulin on cell metabolism and growth. The fundamental research reviewed may be followed by the discovery of molecular receptor defects in clinical syndromes of insulin resistance.     

Structure and function of interleukin-22 and other members of the interleukin-10 family

The IL-10 family of cytokines is comprised of IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and IFN-λs (IL-28A, IL-28B, and IL-29). The IL-10 family members bind to shared class II cytokine receptor chains that associate in various combinations in heterodimeric complexes. Upon interleukin/receptor complex formation, these proteins switch on the Jak/STAT pathway and elicit pleiotropic biological responses whose variety sharply contrasts with their structural similarities. IL-10 family members are involved in several human diseases and health conditions and hence their structural analyses may provide valuable information to design specific therapeutic strategies. In this review, we describe the human interleukin-10 family of cytokines, focusing on their structures and functions, with particular attention given to IL-22 and IL-10. We report on the recently published structures of IL-10 cytokine family members and their complexes with cognate transmembrane and soluble receptors as well as on interleukin physiology and physiopathology.     

Adducin: structure, function and regulation

Adducin is a ubiquitously expressed membrane-skeletal protein localized at spectrin-actin junctions that binds calmodulin and is an in vivo substrate for protein kinase C (PKC) and Rho-associated kinase. Adducin is a tetramer comprised of either alpha/beta or alpha/gamma heterodimers. Adducin subunits are related in sequence and all contain an N-terminal globular head domain, a neck domain and a C-terminal protease-sensitive tail domain. The tail domains of all adducin subunits end with a highly conserved 22-residue myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that has homology to MARCKS protein. Adducin caps the fast-growing ends of actin filaments and also preferentially recruits spectrin to the ends of filaments. Both the neck and the MARCKS-related domains are required for these activities. The neck domain self-associates to form oligomers. The MARCKS-related domain binds calmodulin and contains the major phosphorylation site for PKC. Calmodulin, gelsolin and phosphorylation by the kinase inhibit in vitro activities of adducin involving actin and spectrin. Recent observations suggest a role for adducin in cell motility, and as a target for regulation by Rho-dependent and Ca2+-dependent pathways. Prominent physiological sites of regulation of adducin include dendritic spines of hippocampal neurons, platelets and growth cones of axons.     

The structure and function of platelet-activating factor acetylhydrolases

Platelet-activating factor acetylhydrolases (PAF-AHs, EC 3.1.1.47) constitute a unique and biologically important family of phospholipase A₂s. They are related to neither the well-characterized secretory nor cytosolic PLA₂s, and unlike them do not require Ca²⁺ for catalytic activity. The distinguishing property of PAF-AHs is their unique substrate specificity they act on the phospholipid platelet-activating factor (PAF), and in some cases on proinflammatory polar phospholipids, from which they remove a short acyl moiety – acetyl in the case of PAF – located at the sn-2 position. Because PAF is found both in the plasma and in the cytosol of many tissues, PAF-acetylhydrolases are equally widely distributed in an animal organism. Recent crystallographic studies shed new light on the complex structure-function relationships in PAF-AHs. Received 15 September 1997; received after revision 23 February 1998; accepted 25 February 1998     

Epimerases: structure, function and mechanism

Carbohydrates are ideally suited for molecular recognition. By varying the stereochemistry of the hydroxyl substituents, the simple six-carbon, six-oxygen pyranose ring can exist as 10 different molecules. With the further addition of simple chemical changes, the potential for generating distinct molecular recognition surfaces far exceeds that of amino acids. This ability to control and change the stereochemistry of the hydroxyl substituents is very important in biology. Epimerases can be found in animals, plants and microorganisms where they participate in important metabolic pathways such as the Leloir pathway, which involves the conversion of galactose to glucose-1-phosphate. Bacterial epimerases are involved in the production of complex carbohydrate polymers that are used in their cell walls and envelopes and are recognised as potential therapeutic targets for the treatment of bacterial infection. Several distinct strategies have evolved to invert or epimerise the hydroxyl substituents on carbohydrates. In this review we group epimerisation by mechanism and discuss in detail the molecular basis for each group. These groups include enzymes which epimerise by a transient keto intermediate, those that rely on a permanent keto group, those that eliminate then add a nucleotide, those that break then reform carbon-carbon bonds and those that linearize and cyclize the pyranose ring. This approach highlights the quite different biochemical processes that underlie what is seemingly a simple reaction. What this review shows is that each position on the carbohydrate can be epimerised and that epimerisation is found in all organisms.     

Arabinogalactan-proteins: structure, expression and function

Arabinogalactan-proteins (AGPs) are a family of extensively glycosylated hydroxyproline-rich glycoproteins that are thought to have important roles in various aspects of plant growth and development. After a brief introduction to AGPs highlighting the problems associated with defining and classifying this diverse family of glycoproteins, AGP structure is described in terms of the protein component (including data from molecular cloning), carbohydrate component, processing of AGPs (including recent data on glycosylphosphatidylinositol membrane anchors) and overall molecular shape. Next, the expression of AGPs is examined at several different levels, from the whole plant to the cellular levels, using a variety of experimental techniques and tools. Finally, AGP function is considered. Although the existing functional evidence is not incontrovertible, it does clearly point to roles for AGPs in vegetative, reproductive, and cellular growth and development as well as programmed cell death and social control. In addition and most likely inextricably linked to their functions, AGPs are presumably involved in molecular interactions and cellular signaling at the cell surface. Some likely scenarios are discussed in this context. AGPs also have functions of real or potential commercial value, most notably as emulsifiers in the food industry and as potential immunological regulators for human health. Several important questions remain to be answered with respect to AGPs. Clearly, elucidating the unequivocal functions of particular AGPs and relating these functions to their respective structures and modes of action remain as major challenges in the years ahead.