Juan C. Ferrer

Control of glycogen deposition.

Traditionally, glycogen synthase (GS) has been considered to catalyze the key step of glycogen synthesis and to exercise most of the control over this metabolic pathway. However, recent advances have shown that other factors must be considered. Moreover, the control of glycogen deposition does not follow identical mechanisms in muscle and liver. Glucose must be phosphorylated to promote activation of GS. Glucose‐6‐phosphate (Glc‐6‐P) binds to GS, causing the allosteric activation of the enzyme probably through a conformational rearrangement that simultaneously converts it into a better substrate for protein phosphatases, which can then lead to the covalent activation of GS. The potency of Glc‐6‐P for activation of liver GS is determined by its source, since Glc‐6‐P arising from the catalytic action of glucokinase (GK) is much more effective in mediating the activation of the enzyme than the same metabolite produced by hexokinase I (HK I). As a result, hepatic glycogen deposition from glucose is subject to a system of control in which the ‘controller’, GS, is in turn controlled by GK. In contrast, in skeletal muscle, the control of glycogen synthesis is shared between glucose transport and GS. The characteristics of the two pairs of isoenzymes, liver GS/GK and muscle GS/HK I, and the relationships that they establish are tailored to suit specific metabolic roles of the tissues in which they are expressed. The key enzymes in glycogen metabolism change their intracellular localization in response to glucose. The changes in the intracellular distribution of liver GS and GK triggered by glucose correlate with stimulation of glycogen synthesis. The translocation of GS, which constitutes an additional mechanism of control, causes the orderly deposition of hepatic glycogen and probably represents a functional advantage in the metabolism of the polysaccharide.

Glucokinase regulatory protein is essential for the proper subcellular localisation of liver glucokinase.

Glucokinase (GK), a key enzyme in the glucose homeostatic responses of the liver, changes its intracellular localisation depending on the metabolic status of the cell. Rat liver GK and Xenopus laevis GK, fused to the green fluorescent protein (GFP), concentrated in the nucleus of cultured rat hepatocytes at low glucose and translocated to the cytoplasm at high glucose. Three mutant forms of Xenopus GK with reduced affinity for GK regulatory protein (GKRP) did not concentrate in the hepatocyte nuclei, even at low glucose. In COS‐1 and HeLa cells, a blue fluorescent protein (BFP)‐tagged version of rat liver GK was only able to accumulate in the nucleus when it was co‐expressed with GKRP‐GFP. At low glucose, both proteins concentrated in the nuclear compartment and at high glucose, BFP‐GK translocated to the cytosol while GKRP‐GFP remained in the nucleus. These findings indicate that the presence of and binding to GKRP are necessary and sufficient for the proper intracellular localisation of GK and directly involve GKRP in the control of the GK subcellular distribution.

Intracellular distribution of glycogen synthase and glycogen in primary cultured rat hepatocytes.

Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8 h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.

Structure of the Heme d of Penicillium vitale and Escherichia coli Catalases

A heme d prosthetic group with the configuration of a cis-hydroxychlorin -spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the α--meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.

Muscle glycogen synthase translocates from the cell nucleus to the cytosol in response to glucose

We have studied the intracellular localization of muscular glycogen synthase by fusing the green fluorescent protein (GFP) of the jelly‐fish Aequorea victoria to the N‐terminus of human muscle glycogen synthase (HMGS), and expressing the chimeric protein in C2C12, COS‐1 cells, and primary cultured rat hepatocytes. In contrast to what we have recently found for the hepatic glycogen synthase (Fernández‐Novell et al. (1997) Biochem. J. 321, 227–231), the GFP/HMGS fusion protein is localized to the nucleus of the cell in the absence of glucose, and in the presence of the sugar it is essentially found in the cytosol. Insulin is not required for the translocation of the enzyme.

Crystal Structure of an Archaeal Glycogen Synthase INSIGHTS INTO OLIGOMERIZATION AND SUBSTRATE BINDING OF EUKARYOTIC GLYCOGEN SYNTHASES

Glycogen and starch synthases are retaining glycosyltransferases that catalyze the transfer of glucosyl residues to the non-reducing end of a growing α-1,4-glucan chain, a central process of the carbon/energy metabolism present in almost all living organisms. The crystal structure of the glycogen synthase from Pyrococcus abyssi, the smallest known member of this family of enzymes, revealed that its subunits possess a fold common to other glycosyltransferases, a pair of β/α/β Rossmann fold-type domains with the catalytic site at their interface. Nevertheless, the archaeal enzyme presents an unprecedented homotrimeric molecular arrangement both in solution, as determined by analytical ultracentrifugation, and in the crystal. The C-domains are not involved in intersubunit interactions of the trimeric molecule, thus allowing for movements, likely required for catalysis, across the narrow hinge that connects the N- and C-domains. The radial disposition of the subunits confers on the molecule a distinct triangular shape, clearly visible with negative staining electron microscopy, in which the upper and lower faces present a sharp asymmetry. Comparison of bacterial and eukaryotic glycogen synthases, which use, respectively, ADP or UDP glucose as donor substrates, with the archaeal enzyme, which can utilize both molecules, allowed us to propose the residues that determine glucosyl donor specificity.

Control of liver glycogen synthase activity and intracellular distribution by phosphorylation.

Eukaryotic glycogen synthase activity is regulated by reversible phosphorylation at multiple sites. Of the two GS isoforms found in mammals, the muscle enzyme (muscle glycogen synthase) has received more attention and the relative importance of every known phosphorylation site in the control of its activity and intracellular distribution has been previously addressed. We have analyzed the impact of the dephosphorylation at the homologous sites of the glycogen synthase liver (LGS) isoform. Serine residues at these sites were replaced by non-phosphorylatable alanine residues, singly or in pairs, and the resultant LGS variants were expressed in cultured cells using adenoviral vectors. The sole mutation at site 2 (Ser7) yielded an enzyme that was almost fully active and able to induce glycogen deposition in primary hepatocytes incubated in the absence of glucose and in FTO2B cells, a cell line that does not normally synthesize glycogen. Mutation at site 2 was also sufficient to trigger the aggregation and translocation of LGS from the cytoplasm to the hepatocyte cell cortex in the absence of glucose. However, this redistribution was not observed in hepatocytes incubated without glucose when an additional mutation (E509A), which renders the enzyme inactive, was introduced. This result suggests that LGS translocation is strictly dependent on glycogen synthesis.

Alternative Splicing of the Human Proto-oncogene c-H-ras Renders a New Ras Family Protein That Trafficks to Cytoplasm and Nucleus

The homeobox gene extradenticle (exd) acts as a cofactor of the homeotic genes in the specification of larval patterns during embryogenesis. To study its role in adult patterns, we have generated clones of mutant exd- cells and examined their effect on the different body parts. In some regions, exd- clones exhibit homeotic transformations similar to those produced by known homeotic mutations such as Ultrabithorax (Ubx), labial (lab), spineless-aristapedia (ssa) or Antennapedia (Antp). In other regions, the lack of exd causes novel homeotic transformations producing ectopic eyes and legs. Moreover, exd is also required for functions normally not associated with homeosis, such as the maintenance of the dorsoventral pattern, the specification of subpatterns in adult appendages or the arrangement of bristles in the mesonotum and genitalia. Our findings indicate that exd is critically involved in adult morphogenesis, not only in the homeotic function but also in several other developmental processes.Previously published experiments have shown that the endogenous Dfd gene can be ectopically activated by its own (heat-shock-driven) product in a subset of cells of different segments. This results in the differentiation of maxillary structures like cirri and mouth hooks in places where they normally do not appear, and represents a phenomenon of autocatalysis of homeotic gene function that differs from the normal activation process. We show that this out-of-context activation occurs in cells belonging to the anterior compartments of the three thoracic and the A1 to A8 abdominal segments and that it requires the normal function of the polarity genes wingless (wg) and engrailed (en). The wg product, in addition to that of Dfd, appears to be sufficient to activate the endogenous Dfd gene in many embryonic cells. We have studied the effect of several homeotic genes on Dfd activation and phenotypic expression: Scr, Antp, Ubx and Abd-B repress Dfd both transcriptionally and at the phenotypic level, if their products are in sufficient amounts. The endogenous abd-A gene does not have a noticeable effect, but when it is replaced by an hsp70-abd-A gene, which produces a high and uniform level of expression, the phenotypic expression of Dfd is suppressed. Our results also suggest that the differentiation of cirri is induced by Dfd-expressing cells in non-expressing neighboring cells, and that this interaction occurs across the parasegmental border.During evolution, many animal groups have developed specialised outgrowths of the body wall, limbs or appendages. The type of appendage depends on the identity of the segment where they appear, indicating that the Hox genes contribute to appendage specification. Moreover, work carried out principally in Drosophila has identified the gene products and the mechanisms involved in pattern formation in the appendages. In this essay, we compare the morphogenetic processes in the appendages and the body wall; the function of the Hox genes and the response to the signalling molecules involved in local patterning. We speculate that, although the basic mechanisms are similar, there are significant differences in the manner the body trunk and appendages respond to them.[ES] La pared celular es un elemento morfogenetico esencial que determina la forma final de las celulas y que las protege contra la lisis. En S. pombe esta esta constituida por ? y s-glucano y manoproteinas y tanto la sintesis como remodelacion de su estructura requiere de diferentes enzimas estrictamente reguladas. En S. pombe existe poca informacion de como se lleva a cabo la incorporacion del material de membrana y sobre la regulacion de las enzimas implicadas en la sintesis y remodelacion de la pared celular por los mecanismos de transporte vesicular. Para abordar el estudio de como el trafico vesicular mediado por clatrina afecta a la morfogenesis de S. pombe y en particular cual es su papel en la regulacion de la sintesis de la pared celular se ha analizado el papel tanto de la clatrina, mediante el analisis de diferentes mutantes de la cadena ligera de la clatrina, como el del adaptador AP-2, que interviene en el proceso de endocitosis mediada por clatrina. Se ha demostrado que la delecion de la cadena ligera de la clatrina resulta letal para las celulas de S. pombe y que esta letalidad se rescata al incubar las celulas en un medio suplementado con sorbitol. En este caso aunque las celulas pueden sobrevivir poseen graves defectos morfologicos, en crecimiento, en trafico vesicular, en desarrollo sexual, etc. Se ha podido comprobar que la ausencia de Clc1p afecta drasticamente a la estabilidad de Chc1p hecho que hace que, a diferencia de otros organismos, la supervivencia de S. pombe sea mas dependiente de la presencia clatrina. Ademas se ha demostrado que la letalidad causada por la ausencia de Clc1p se debe principalmente a defectos graves en la sintesis de la pared celular que afectan directamente a la sintesis del glucano. Los resultados obtenidos muestran que una reduccion en la cantidad de clatrina causa un leve impacto en el transporte vesicular en general y en otros procesos y elementos biologicos, pero afecta gravemente a la secrecion de enzimas de sintesis/remodelacion de la pared celular, como las s(1,3)glucan sintasa y endoglucanasas. En cuanto al complejo adaptador AP-2 se ha comprobado, que a diferencia de lo que se conoce hasta el momento en otros organismos unicelulares, este forma un complejo con la clatrina y se ha demostrado que tiene un papel en la endocitosis general de S. pombe. Asi mismo se ha descubierto que AP-2 puede estar interviniendo en la sintesis de la pared celular ya que su ausencia afecta a la actividad s-glucan sintasa y hace que S. pombe sea hiper-sensible a compuestos que afectan a la integridad de la pared celular.We characterized a novel protein of the Ras family, p19 (H-RasIDX). The c-H-ras proto-oncogene undergoes alternative splicing of the exon termed IDX. We show that the alternative p19 mRNA is stable and as abundant as p21 (p21 H-Ras4A) mRNA in all of the human tissues and cell lines tested. IDX is spliced into stable mRNA in different mammalian species, which present a high degree of nucleotide conservation. Both the endogenous and the transiently expressed p19 protein are detected in COS-1 and HeLa cells and show nuclear diffuse and speckled patterns as well as cytoplasmic localization. In yeast two-hybrid assays, p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multiprotein complexes in different signaling pathways. This observation suggests that p19 and p21 play differential and complementary roles in the cell.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.A. G. G. thanks Ramon Areces Foundation for a grant. J. C. thanks NIH-CA24487 for financial support.Ministerio de Educacion y Ciencia and grant S-0505/MAT-0283 from Comunidad Autonoma de Madrid to M.S. and by an Institutional grant from Fundacion Ramon Areces to the Centro de Biologia Molecular “Severo Ochoa”We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.The pannier (pnr) gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. We report some novel aspects of the regulation and function of pnr during embryogenesis. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but we find that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, we show that pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila.10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 pageBy using a hsp70-Ubx fusion gene, we have ectopically expressed a Ubx product in the embryonic head primordia and studied the developmental effects on the larval head. We find that after high and persistent levels of Ubx product, the head is replaced by three (C1, C2 and C3) abdominal-like denticle belts. The C2 and C3 belts are the homeotic transformations of parasegments 1 and 2, respectively, while the C1 belt probably derives from the transformation and subsequent fusion of the most anterior procephalic primordia. On the basis of their response to the Ubx product and other arguments, we propose that the larval head is made of two genetically distinct components; one is the procephalon and the anterior region of the mandibular lobe, and the other is part of the parasegmental trunk and includes parasegments 1 and 2. Our results also indicate that most or all the larval head structures derive from precursor cells of ventral origin.The Iroquois (Iro) family of genes are found in nematodes, insects and vertebrates. They usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Iro genes function early in development to specify the identity of diverse territories of the body, such as the dorsal head and dorsal mesothorax of Drosophila and the neural plate of Xenopus. In some aspects they act in the same way as classical selector genes, but they display specific properties that place them into a category of their own. Later in development in both Drosophila and vertebrates, the Iro genes function again to subdivide those territories into smaller domains.The pannier (pnr) gene encodes a GATA transcription factor and acts in several developmental processes in Drosophila, including embryonic dorsal closure, specification of cardiac cells and bristle determination. We show that pnr is expressed in the mediodorsal parts of thoracic and abdominal segments of embryos, larvae and adult flies. Its activity confers cells with specific adhesion properties that make them immiscible with non-expressing cells. Thus there are two genetic domains in the dorsal region of each segment: a medial (MED) region where pnr is expressed and a lateral (LAT) region where it is not. The homeobox gene iroquois (iro) is expressed in the LAT region. These regions are not formed by separate polyclones of cells, but are defined topographically. We show that ectopic pnr in the wing induces MED thoracic development, indicating that pnr specifies the identity of the MED regions. Correspondingly, when pnr is removed from clones of cells in the MED domain, they sort out and apparently adopt the LAT fate. We propose that (1) the subdivision into MED and LAT regions is a general feature of the Drosophila body plan and (2) pnr is the principal gene responsible for this subdivision. We argue that pnr acts like a classical selector gene but differs in that its expression is not propagated through cell divisions.We have developed a specific polyclonal antibody that recognizes the protein products of the abdominal-A (abd-A) gene, a member of the bithorax complex of Drosophila. The normal expression domain extends from parasegments 7 to 13, in good correspondence with previous genetic and molecular results. However, while the anterior border of expression is precisely demarcated by a parasegmental boundary, the posterior border does not coincide with a lineage boundary. Within the normal domain, the expression of abd-A shows intrametameric modulation; the amount of product is higher in posterior compartments and in the most anterior cells of the anterior compartments and then gradually decreases. We have examined the effect on abd-A expression of a number of mutations, some mapping within and others outside the abd-A transcription unit. Those mapping to the transcription unit eliminate or severely reduce the amount of abd-A antigen, while those mapping outside produce an abnormal distribution of abd-A protein. Finally, we show that the abd-A gene is down-regulated in part of the Abdominal-B (Abd-B) domain, precisely in those regions where the Abd-B gene is expressed at high levels.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.The effect of the anti-tumoral drug lauryl gallate on the infectivity of the African swine fever virus among other DNA (Herpes simplex and Vaccinia) and RNA (Influenza, Porcine transmissible gastroenteritis and Sindbis) viruses, involved in animal and human diseases, is analyzed. Viral production was strongly inhibited in different cell lines at non-toxic concentrations of the drug (1-10 μM), reducing the titres from 3 to more than 5 log. units depending on the multiplicity of infection. In our model system (African swine fever virus in Vero cells), the addition of the drug 1 h before virus adsorption, completely abolished virus productivity in a one-step growth virus cycle. Interestingly, no inhibitory effect was observed when lauryl gallate was added after 5 to 8 hpi. Both cellular and viral DNA synthesis and late viral transcription were inhibited by the drug, but, however, the early viral protein synthesis and the virus-mediated increasing of p53 remained unaffected. Activation of the apoptotic effector caspase-3 was not detected after lauryl gallate treatment of Vero cells, and, furthermore, the presence of the drug abrogated the activation of this protease induced by the virus infection. The overall results likely indicate that a cellular factor/function might be the target of the antiviral action of alkyl gallates.Tesis Doctoral presentada por Eduardo Rodenas Martinez en el Centro Andaluz de Biologia del Desarrollo, centro mixto CSIC-UPO.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.

Intracellular distribution of hepatic glucokinase and glucokinase regulatory protein during the fasted to refed transition in rats

We have studied the intracellular distribution in vivo of glucokinase (GK) and glucokinase regulatory protein (GKRP) in livers of fasted and refed rats, using specific antibodies against both proteins and laser confocal fluorescence microscopy. GK was found predominantly in the nucleus of hepatocytes from starved rats. GK was translocated to the cytoplasm in livers of 1‐ and 2‐h refed animals, but returned to the nucleus after 4 h. GKRP concentrated in the hepatocyte nuclei and its distribution did not change upon refeeding. These results show that, in physiological conditions, GKRP is present predominantly in the nuclei of hepatocytes and that the translocation of hepatic GK from and to the nucleus is operative in vivo.

Liver glycogen synthase but not the muscle isoform differentiates between glucose 6-phosphate produced by glucokinase or hexokinase.

Using adenovirus-mediated gene transfer into FTO-2B cells, a rat hepatoma cell line, we have overexpressed hexokinase I (HK I), glucokinase (GK), liver glycogen synthase (LGS), muscle glycogen synthase (MGS), and combinations of each of the two glucose-phosphorylating enzymes with each one of the GS isoforms. FTO-2B cells do not synthesize glycogen even when incubated with high doses of glucose. Adenovirus-induced overexpression of HK I and/or LGS, two enzymes endogenously expressed by these cells, did not produce a significant increase in the levels of active GS and the total glycogen content. In contrast, GK overexpression led to the glucose-dependent activation of endogenous or overexpressed LGS and to the accumulation of glycogen. Similarly overexpressed MGS was efficiently activated by the glucose-6-phosphate (Glc-6-P) produced by either endogenous or overexpressed HK I and by overexpressed GK. These results indicate the existence of at least two pools of Glc-6-P in the cell, one of them is accessible to both isoforms of GS and is replenished by the action of GK, whereas LGS is excluded from the cellular compartment where the Glc-6-P produced by HK I is directed. These findings are interpreted in terms of the metabolic role that the two pairs of enzymes, HK I-MGS in the muscle and GK-LGS in the hepatocyte, perform in their respective tissues.