Mario Pantoja

Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex.

The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, we used a model organism Drosophila melanogaster and performed genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-β and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought.

Agrobacterium type IV secretion is a two‐step process in which export substrates associate with the virulence protein VirJ in the periplasm

Type IV secretion systems are virulence determinants in many bacteria and share extensive homology with many conjugal transfer systems. Although type IV systems and their homologues have been studied widely, the mechanism by which substrates are secreted remains unclear. In Agrobacterium, we show that type IV secretion substrates that lack signal peptides form a soluble complex in the periplasm with the virulence protein VirJ. Additionally, these proteins co‐precipitate with constituents of the type IV transporter: the VirB pilus and the VirD4 protein. Our findings suggest that the substrate proteins localized to the periplasm may associate with the pilus in a manner that is mediated by VirJ, and suggest a two‐step process for type IV secretion in Agrobacterium. Our analyses of protein–protein interactions in a variety of mutant backgrounds indicate that substrates are probably secreted independently of one another.

Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease. Studies in Drosophila showed that genetic increase of the levels of the bioactive sphingolipid sphingosine-1-phosphate (S1P) or delivery of 2-acetyl-5-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, suppresses dystrophic muscle degeneration. In the dystrophic mouse (mdx), upregulation of S1P by THI increases regeneration and muscle force. S1P can act as a ligand for S1P receptors and as a histone deacetylase (HDAC) inhibitor. Because Drosophila has no identified S1P receptors and DMD correlates with increased HDAC2 levels, we tested whether S1P action in muscle involves HDAC inhibition. Here we show that beneficial effects of THI treatment in mdx mice correlate with significantly increased nuclear S1P, decreased HDAC activity and increased acetylation of specific histone residues. Importantly, the HDAC2 target microRNA genes miR-29 and miR-1 are significantly upregulated, correlating with the downregulation of the miR-29 target Col1a1 in the diaphragm of THI-treated mdx mice. Further gene expression analysis revealed a significant THI-dependent decrease in inflammatory genes and increase in metabolic genes. Accordingly, S1P levels and functional mitochondrial activity are increased after THI treatment of differentiating C2C12 cells. S1P increases the capacity of the muscle cell to use fatty acids as an energy source, suggesting that THI treatment could be beneficial for the maintenance of energy metabolism in mdx muscles.

Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

BackgroundPresently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice.MethodsWe treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles.ResultsShort-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer-term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular injection of S1P increased the number of Myf5nlacz/+ positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function.ConclusionsThese data show that S1P is beneficial for muscle regeneration and functional gain in dystrophic mice, and that THI, or other pharmacological agents that raise S1P levels systemically, may be developed into an effective treatment for improving muscle function and reducing the pathology of DMD.

Genetic elevation of Sphingosine 1-phosphate suppresses dystrophic muscle phenotypes in Drosophila

Duchenne muscular dystrophy is a lethal genetic disease characterized by the loss of muscle integrity and function over time. Using Drosophila, we show that dystrophic muscle phenotypes can be significantly suppressed by a reduction of wunen, a homolog of lipid phosphate phosphatase 3, which in higher animals can dephosphorylate a range of phospholipids. Our suppression analyses include assessing the localization of Projectin protein, a titin homolog, in sarcomeres as well as muscle morphology and functional movement assays. We hypothesize that wunen-based suppression is through the elevation of the bioactive lipid Sphingosine 1-phosphate (S1P), which promotes cell proliferation and differentiation in many tissues, including muscle. We confirm the role of S1P in suppression by genetically altering S1P levels via reduction of S1P lyase (Sply) and by upregulating the serine palmitoyl-CoA transferase catalytic subunit gene lace, the first gene in the de novo sphingolipid biosynthetic pathway and find that these manipulations also reduce muscle degeneration. Furthermore, we show that reduction of spinster (which encodes a major facilitator family transporter, homologs of which in higher animals have been shown to transport S1P) can also suppress dystrophic muscle degeneration. Finally, administration to adult flies of pharmacological agents reported to elevate S1P signaling significantly suppresses dystrophic muscle phenotypes. Our data suggest that localized intracellular S1P elevation promotes the suppression of muscle wasting in flies.

Dystroglycan and mitochondrial ribosomal protein L34 regulate differentiation in the Drosophila eye.

Mutations that diminish the function of the extracellular matrix receptor Dystroglycan (DG) result in muscular dystrophies, with associated neuronal migration defects in the brain and mental retardation e.g. Muscle Eye Brain Disease. To gain insight into the function of DG in the nervous system we initiated a study to examine its contribution to development of the eye of Drosophila melanogaster. Immuno-histochemistry showed that DG is concentrated on the apical surface of photoreceptors (R) cells during specification of cell-fate in the third instar larva and is maintained at this location through early pupal stages. In point mutations that are null for DG we see abortive R cell elongation during differentiation that first appears in the pupa and results in stunted R cells in the adult. Overexpression of DG in R cells results in a small but significant increase in their size. R cell differentiation defects appear at the same stage in a deficiency line Df(2R)Dg248 that affects Dg and the neighboring mitochondrial ribosomal gene, mRpL34. In the adult, these flies have severely disrupted R cells as well as defects in the lens and ommatidia. Expression of an mRpL34 transgene rescues much of this phenotype. We conclude that DG does not affect neuronal commitment but functions R cell autonomously to regulate neuronal elongation during differentiation in the pupa. We discuss these findings in view of recent work implicating DG as a regulator of cell metabolism and its genetic interaction with mRpL34, a member of a class of mitochondrial genes essential for normal metabolic function.

The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

BackgroundDystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys) WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro.ResultsWe now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required.ConclusionBased on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.

Drosophila as a starting point for developing therapeutics for the rare disease Duchenne Muscular Dystrophy

Progress into developing therapeutics for rare diseases can be accelerated for those diseases that can be modeled in genetically tractable organisms. Here we comment on one disease, Duchenne Muscular Dystrophy (DMD), modeled in Drosophila that brought together disparate lines of research toward the goal of developing a therapeutic. Though the bioactive lipid sphingosine 1-phosphate (S1P) has been implicated in many anabolic processes in many cell types and tissues, including muscle, this work confirmed the therapeutic potential of assessing this pathway for DMD. Genetic dissection of sphingolipid metabolism showed the suppression of muscle structural and functional defects in flies. Moreover, improvement of muscle defects using known pharmacological agents that raise S1P levels in vivo highlight the potential of Drosophila as a drug-screening tool for DMD. We and others have extended S1P studies into the mouse model of DMD and have shown a partial amelioration of symptoms associated with DMD. Translation of this work to mammals makes the sphingolipid metabolism pathway a promising target for further drug development that may benefit the human condition.

Sequential removal of monosaccharides from the reducing end of oligosaccharides. I. A reaction between hydrazine and sugars having a glycosidic substituent on a carbon atom adjacent to the carbonyl group

Abstract Hydrazine reacts smoothly with sugars having a glycosidic substituent when the glycosyl moiety is located on a carbon atom adjacent to an aldehyde or keto group, resulting in cleavage of the glycosidic linkage. In excess hydrazine, the released glycoside forms a hydrazone from which the reducing sugar may be recovered in high yields.