Biomedical subjects

Graeme M Birdsey

Publications and source records attributed to Graeme M Birdsey.

8 recordsLinked to original sources

PubMed2005

Endothelial intercellular adhesion molecule (ICAM)-2 regulates angiogenesis.

Endothelial junctions maintain endothelial integrity and vascular homeostasis. They modulate cell trafficking into tissues, mediate cell-cell contact and regulate endothelial survival and apoptosis. Junctional adhesion molecules such as vascular endothelial (VE)-cadherin and CD31/platelet endothelial cell adhesion molecule (PECAM) mediate contact between adjacent endothelial cells and regulate leukocyte transmigration and angiogenesis. The leukocyte adhesion molecule intercellular adhesion molecule 2 (ICAM-2) is expressed at the endothelial junctions. In this study we demonstrate that endothelial ICAM-2 also mediates angiogenesis. Using ICAM-2-deficient mice and ICAM-2-deficient endothelial cells, we show that the lack of ICAM-2 expression results in impaired angiogenesis both in vitro and in vivo. We show that ICAM-2 supports homophilic interaction, and that this may be involved in tube formation. ICAM-2-deficient cells show defective in vitro migration, as well as increased apoptosis in response to serum deprivation, anti-Fas antibody, or staurosporine. ICAM-2 signaling in human umbilical vein endothelial cells (HUVECs) was found to activate the small guanosine triphosphatase (GTPase) Rac, which is required for endothelial tube formation and migration. These data indicate that ICAM-2 may regulate angiogenesis via several mechanisms including survival, cell migration, and Rac activation. Our findings identify a novel pathway regulating angiogenesis through ICAM-2 and a novel mechanism for Rac activation during angiogenesis.

Animals↗

PubMed2005

Peroxisomal import of human alanine:glyoxylate aminotransferase requires ancillary targeting information remote from its C terminus.

Although human alanine:glyoxylate aminotransferase (AGT) is imported into peroxisomes by a Pex5p-dependent pathway, the properties of its C-terminal tripeptide (KKL) are unlike those of any other type 1 peroxisomal targeting sequence (PTS1). We have previously suggested that AGT might possess ancillary targeting information that enables its unusual PTS1 to work. In this study, we have attempted to locate this information and to determine whether or not it is a characteristic of all vertebrate AGTs. Using the two-hybrid system, we show that human AGT interacts with human Pex5p in mammalian cells, but not yeast cells. Using (immuno)fluorescence microscopic analysis of the distribution of various constructs expressed in COS cells, we show the following. 1) The putative ancillary peroxisomal targeting information (PTS1A) in human AGT is located entirely within the smaller C-terminal structural domain of 110 amino acids, with the sequence between Val-324 and Ile-345 being the most likely candidate region. 2) The PTS1A is present in all mammalian AGTs studied (human, rat, guinea pig, rabbit, and cat), but not amphibian AGT (Xenopus). 3) The PTS1A is necessary for peroxisomal import of human, rabbit, and cat AGTs, but not rat and guinea pig AGTs. We speculate that the internal PTS1A of human AGT works in concert with the C-terminal PTS1 by interacting with Pex5p indirectly with the aid of a yet-to-be-identified mammal-specific adaptor molecule. This interaction might reshape the tetratricopeptide repeat domain allosterically, enabling it to accept KKL as a functional PTS1.

Amino Acid Sequence↗

PubMed2005

A comparative analysis of the evolutionary relationship between diet and enzyme targeting in bats, marsupials and other mammals.

The subcellular distribution of the enzyme alanine:glyoxylate aminotransferase (AGT) in the livers of different mammals appears to be related to their natural diets. Thus, AGT tends to be mitochondrial in carnivores, peroxisomal in herbivores, and both mitochondrial and peroxisomal in omnivores. To what extent this relationship is an incidental consequence of phylogenetic structure or an evolutionarily meaningful adaptive response to changes in dietary selection pressure is unknown. In order to distinguish between these two possibilities, we have determined the subcellular distribution of AGT in the livers of 22 new mammalian species, including members of three orders not studied before. In addition, we have analysed the statistical relationship between AGT distribution and diet in all 77 mammalian species, from 12 different orders, for which the distribution is currently known. Our analysis shows that there is a highly significant correlation between AGT distribution and diet, independent of phylogeny. This finding is compatible with the suggestion that the variable intracellular targeting of AGT is an adaptive response to episodic changes in dietary selection pressure. To our knowledge, this is the first example of such a response being manifested at the molecular and cellular levels across the breadth of Mammalia.

Alanine Transaminase↗

PubMed2004

Differential enzyme targeting as an evolutionary adaptation to herbivory in carnivora.

Not all members of the order Carnivora are carnivorous. Some are omnivorous, and a few, such as the giant panda, Ailuropoda melanoleuca, are almost exclusively herbivorous. Although a number of adaptations to increased plant-eating are recognized within Carnivora, few have been studied at the molecular level. One molecular adaptation to diet that is spread widely across Mammalia is the differential intracellular targeting of the intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT), which tends to be mitochondrial in carnivores, peroxisomal in herbivores, and both mitochondrial and peroxisomal in omnivores. In the present study, we have analyzed the targeting of AGT in Carnivora in relation to species' natural diets. We show not only that there has been an adaptive shift in AGT targeting from the mitochondrion toward the peroxisome as diets have shifted from being mainly carnivorous to ones that are more omnivorous and herbivorous but also that in one lineage, namely that of the giant panda, there is evidence for positive selection pressure at the molecular level on the AGT mitochondrial targeting sequence to decrease its efficiency, thereby allowing more AGT to be targeted to the peroxisomes.

Adaptation, Biological↗

PubMed2003

Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels, VEGF expression, and cell phenotype.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Elevated levels of circulating ADMA correlate with various cardiovascular pathologies less is known about the cellular effects of altered DDAH activity. We modified DDAH activity in cells and measured the changes in ADMA levels, morphological phenotypes on Matrigel, and expression of vascular endothelial growth factor (VEGF). DDAH over-expressing ECV304 cells secreted less ADMA and when grown on Matrigel had enhanced tube formation compared to untransfected cells. VEGF mRNA levels were 2.1-fold higher in both ECV304 and murine endothelial cells (sEnd.1) over-expressing DDAH. In addition the DDAH inhibitor, S-2-amino-4(3-methylguanidino)butanoic acid (4124W 1mM), markedly reduced human umbilical vein endothelial cell tube formation in vitro. We have found that upregulating DDAH activity lowers ADMA levels, increases the levels of VEGF mRNA in endothelial cells, and enhances tube formation in an in vitro model, whilst blockade of DDAH reduces tube formation.

Amidohydrolases↗

PubMed2003

Correction of an enzyme trafficking defect in hereditary kidney stone disease in vitro.

In normal human hepatocytes, the intermediary-metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is located within the peroxisomes. However, in approx. one-third of patients suffering from the hereditary kidney stone disease primary hyperoxaluria type 1, AGT is mistargeted to the mitochondria. AGT mistargeting results from the synergistic interaction between a common P11L (Pro11-->Leu) polymorphism and a disease-specific G170R mutation. The polymorphism generates a functionally weak mitochondrial targeting sequence, the efficiency of which is increased by the mutation. The two substitutions together, but not in isolation, inhibit AGT dimerization, highlighting the different structural requirements of the peroxisomal and mitochondrial protein-import machineries. In the present study, we show that treatments known to increase the stability of proteins non-specifically (i.e. lowering the temperature from 37 to 30 degrees C or by the addition of glycerol) completely normalize the intracellular targeting of mutant AGT expressed in transfected COS cells. On the other hand, treatments known to decrease protein stability (e.g. increasing the temperature from 37 to 42 degrees C) exacerbate the targeting defect. Neither of the treatments affects the relative efficiencies of the peroxisomal and mitochondrial protein-import pathways intrinsically. Results are discussed in the light of the known structural requirements of the two protein trafficking pathways and the formulation of possible treatment strategies for primary hyperoxaluria type 1.

Alleles↗

PubMed2003

Fungal hydrogenosomes contain mitochondrial heat-shock proteins.

At least three groups of anaerobic eukaryotes lack mitochondria and instead contain hydrogenosomes, peculiar organelles that make energy and excrete hydrogen. Published data indicate that ciliate and trichomonad hydrogenosomes share common ancestry with mitochondria, but the evolutionary origins of fungal hydrogenosomes have been controversial. We have now isolated full-length genes for heat shock proteins 60 and 70 from the anaerobic fungus Neocallimastix patriciarum, which phylogenetic analyses reveal share common ancestry with mitochondrial orthologues. In aerobic organisms these proteins function in mitochondrial import and protein folding. Homologous antibodies demonstrated the localization of both proteins to fungal hydrogenosomes. Moreover, both sequences contain amino-terminal extensions that in heterologous targeting experiments were shown to be necessary and sufficient to locate both proteins and green fluorescent protein to the mitochondria of mammalian cells. This finding, that fungal hydrogenosomes use mitochondrial targeting signals to import two proteins of mitochondrial ancestry that play key roles in aerobic mitochondria, provides further strong evidence that the fungal organelle is also of mitochondrial ancestry. The extraordinary capacity of eukaryotes to repeatedly evolve hydrogen-producing organelles apparently reflects a general ability to modify the biochemistry of the mitochondrial compartment.

Amino Acid Sequence↗

PubMed2003

Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease.

The pyridoxal-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria in patients with the hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) due to the synergistic interaction between a common Pro(11)Leu polymorphism and a PH1-specific Gly(170)Arg mutation. The kinetic partitioning of newly synthesised AGT between peroxisomes and mitochondria is determined by the combined effects of (1) the generation of cryptic mitochondrial targeting information, and (2) the inhibition of AGT dimerization. The crystal structure of AGT has recently been solved, allowing the effects of the various polymorphisms and mutations to be rationalised in terms of AGT's three-dimensional conformation. Procedures that increase dimer stability and/or increase the rate of dimer formation have potential in the formulation of novel strategies to treat this otherwise intractable life-threatening disease.

Alanine↗