Subcellular localization directed by specific targeting motifs is an emerging theme for regulatin... more Subcellular localization directed by specific targeting motifs is an emerging theme for regulating signal transduction pathways. For cAMP-dependent protein kinase (PKA), this is achieved primarily by its association with A-kinase-anchoring proteins (AKAPs). Dual specificity AKAP1, (D-AKAP1) binds to both type I and type II regulatory subunits and has two NH2-terminal (N0 and N1) and two COOH-terminal (C1 and C2) splice variants (. J. Biol. Chem. 272:8057). Here we report that the splice variants of D-AKAP1 are expressed in a tissue-specific manner with the NH2-terminal motifs serving as switches to localize D-AKAP1 at different sites. Northern blots showed that the N1 splice is expressed primarily in liver, while the C1 splice is predominant in testis. The C2 splice shows a general expression pattern. Microinjecting expression constructs of D-AKAP1(N0) epitope-tagged at either the NH2 or the COOH terminus showed their localization to the mitochondria based on immunocytochemistry. De...
We found that targeting cholesterol with beta-cyclodextrin (bCD) and its derivatives disrupted si... more We found that targeting cholesterol with beta-cyclodextrin (bCD) and its derivatives disrupted signal transduction between PI3K and AKT, attenuating AKT pro-survival signals. In their absence, 2-deoxyglucose (2DG) caused anti-apoptotic protein Mcll to dissociate from pro-apoptotic Bak at mitochondria. Normally Bak is sequestered by its inhibitory associations with Mcll and Bcl-xL, and only when Bak is released from both, is it free to form oligomers through which cytochrome c can escape into the cytosol. Thus an addition of a bcl-2 antagonist dissociates Bak from Bcl-xL, triggering cytochrome c release and inducing apoptosis. 2DG-bCD can also sensitize type II cancer cells for TRAIL-mediated apoptosis.
Chronic activation of mammalian target of rapamycin complex 1 (mTORC1) and p70 S6 kinase (S6K) in... more Chronic activation of mammalian target of rapamycin complex 1 (mTORC1) and p70 S6 kinase (S6K) in response to hypernutrition contributes to obesity-associated metabolic pathologies, including hepatosteatosis and insulin resistance. Sestrins are stress-inducible proteins that activate AMP-activated protein kinase (AMPK) and suppress mTORC1-S6K activity, but their role in mammalian physiology and metabolism has not been investigated. We show that Sestrin2--encoded by the Sesn2 locus, whose expression is induced upon hypernutrition--maintains metabolic homeostasis in liver of obese mice. Sesn2 ablation exacerbates obesity-induced mTORC1-S6K activation, glucose intolerance, insulin resistance, and hepatosteatosis, all of which are reversed by AMPK activation. Furthermore, concomitant ablation of Sesn2 and Sesn3 provokes hepatic mTORC1-S6K activation and insulin resistance even in the absence of nutritional overload and obesity. These results demonstrate an important homeostatic function for the stress-inducible Sestrin protein family in the control of mammalian lipid and glucose metabolism.
Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a reg... more Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40Â % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200Â nm) than in WT mice (200-350Â nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47Â % increase in DCV/DC ratio, implying vesicle swelling due to increased ...
Cyclic AMP (cAMP), acting via protein kinase A (PKA), regulates many cellular responses but the r... more Cyclic AMP (cAMP), acting via protein kinase A (PKA), regulates many cellular responses but the role of mitochondria in such responses is poorly understood. To define such roles, we used quantitative proteomic analysis of mitochondria-enriched fractions and performed functional and morphologic studies of wild-type (WT) and kin- (PKA-null) murine S49 lymphoma cells. Basally, 75 proteins significantly differed in abundance between WT and kin- S49 cells. WT, but not kin-, S49 cells incubated with the cAMP analog 8-(4-Chlorophenylthio)adenosine cAMP (CPT-cAMP) for 16h have: a) increased expression of mitochondria-related genes and proteins, including ones in pathways of branched chain amino acid (BCAA) and fatty acid metabolism, and b) increased maximal capacity of respiration on branched chain keto acids and fatty acids. CPT-cAMP also regulates the cellular rate of ATP-utilization, as the rates of both ATP-linked respiration and proton efflux are decreased in WT but not kin- cells. CPT...
Comparative studies of structure related to function offer a promising means of understanding the... more Comparative studies of structure related to function offer a promising means of understanding the significance of differences in cytoarchitecture. Mitochondrial crista structure is linked tightly to mitochondrial function. Non-foveal cone photoreceptors of primates contain considerably more inner segment mitochondria and have higher oxidative enzyme activity than do rods. In addition, it is suggested that light-adapted cones have a higher aerobic ATP demand than light-adapted rods. Therefore, we investigated the oxidative metabolism and three-dimensional membrane architecture of mouse rod and cone inner segment mitochondria. We determined the number, size, cytochrome c oxidase (CO) reactivity, and membrane architecture of rod and middle wavelength-sensitive (M) cone inner segment mitochondria from 21 day old light-adapted C57BL/6 mice using conventional electron microscopy and the three-dimensional approach of single- and double-tilt electron microscope tomography. Fourteen differen...
Abnormal structure and function of astrocytes have been observed within the lamina cribrosa regio... more Abnormal structure and function of astrocytes have been observed within the lamina cribrosa region of the optic nerve head (ONH) in glaucomatous neurodegeneration. Glutamate excitotoxicity-mediated mitochondrial alteration has been implicated in experimental glaucoma. However, the relationships among glutamate excitotoxicity, mitochondrial alteration and ONH astrocytes in the pathogenesis of glaucoma remain unknown. We found that functional N-methyl-d-aspartate (NMDA) receptors (NRs) are present in human ONH astrocytes and that glaucomatous human ONH astrocytes have increased expression levels of NRs and the glutamate aspartate transporter. Glaucomatous human ONH astrocytes exhibit mitochondrial fission that is linked to increased expression of dynamin-related protein 1 and its phosphorylation at Serine 616. In BAC ALDH1L1 eGFP or Thy1-CFP transgenic mice, NMDA treatment induced axon loss as well as hypertrophic morphology and mitochondrial fission in astrocytes of the glial lamina....
Subcellular localization directed by specific targeting motifs is an emerging theme for regulatin... more Subcellular localization directed by specific targeting motifs is an emerging theme for regulating signal transduction pathways. For cAMP-dependent protein kinase (PKA), this is achieved primarily by its association with A-kinase-anchoring proteins (AKAPs). Dual specificity AKAP1, (D-AKAP1) binds to both type I and type II regulatory subunits and has two NH2-terminal (N0 and N1) and two COOH-terminal (C1 and C2) splice variants (. J. Biol. Chem. 272:8057). Here we report that the splice variants of D-AKAP1 are expressed in a tissue-specific manner with the NH2-terminal motifs serving as switches to localize D-AKAP1 at different sites. Northern blots showed that the N1 splice is expressed primarily in liver, while the C1 splice is predominant in testis. The C2 splice shows a general expression pattern. Microinjecting expression constructs of D-AKAP1(N0) epitope-tagged at either the NH2 or the COOH terminus showed their localization to the mitochondria based on immunocytochemistry. De...
We found that targeting cholesterol with beta-cyclodextrin (bCD) and its derivatives disrupted si... more We found that targeting cholesterol with beta-cyclodextrin (bCD) and its derivatives disrupted signal transduction between PI3K and AKT, attenuating AKT pro-survival signals. In their absence, 2-deoxyglucose (2DG) caused anti-apoptotic protein Mcll to dissociate from pro-apoptotic Bak at mitochondria. Normally Bak is sequestered by its inhibitory associations with Mcll and Bcl-xL, and only when Bak is released from both, is it free to form oligomers through which cytochrome c can escape into the cytosol. Thus an addition of a bcl-2 antagonist dissociates Bak from Bcl-xL, triggering cytochrome c release and inducing apoptosis. 2DG-bCD can also sensitize type II cancer cells for TRAIL-mediated apoptosis.
Chronic activation of mammalian target of rapamycin complex 1 (mTORC1) and p70 S6 kinase (S6K) in... more Chronic activation of mammalian target of rapamycin complex 1 (mTORC1) and p70 S6 kinase (S6K) in response to hypernutrition contributes to obesity-associated metabolic pathologies, including hepatosteatosis and insulin resistance. Sestrins are stress-inducible proteins that activate AMP-activated protein kinase (AMPK) and suppress mTORC1-S6K activity, but their role in mammalian physiology and metabolism has not been investigated. We show that Sestrin2--encoded by the Sesn2 locus, whose expression is induced upon hypernutrition--maintains metabolic homeostasis in liver of obese mice. Sesn2 ablation exacerbates obesity-induced mTORC1-S6K activation, glucose intolerance, insulin resistance, and hepatosteatosis, all of which are reversed by AMPK activation. Furthermore, concomitant ablation of Sesn2 and Sesn3 provokes hepatic mTORC1-S6K activation and insulin resistance even in the absence of nutritional overload and obesity. These results demonstrate an important homeostatic function for the stress-inducible Sestrin protein family in the control of mammalian lipid and glucose metabolism.
Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a reg... more Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40Â % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200Â nm) than in WT mice (200-350Â nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47Â % increase in DCV/DC ratio, implying vesicle swelling due to increased ...
Cyclic AMP (cAMP), acting via protein kinase A (PKA), regulates many cellular responses but the r... more Cyclic AMP (cAMP), acting via protein kinase A (PKA), regulates many cellular responses but the role of mitochondria in such responses is poorly understood. To define such roles, we used quantitative proteomic analysis of mitochondria-enriched fractions and performed functional and morphologic studies of wild-type (WT) and kin- (PKA-null) murine S49 lymphoma cells. Basally, 75 proteins significantly differed in abundance between WT and kin- S49 cells. WT, but not kin-, S49 cells incubated with the cAMP analog 8-(4-Chlorophenylthio)adenosine cAMP (CPT-cAMP) for 16h have: a) increased expression of mitochondria-related genes and proteins, including ones in pathways of branched chain amino acid (BCAA) and fatty acid metabolism, and b) increased maximal capacity of respiration on branched chain keto acids and fatty acids. CPT-cAMP also regulates the cellular rate of ATP-utilization, as the rates of both ATP-linked respiration and proton efflux are decreased in WT but not kin- cells. CPT...
Comparative studies of structure related to function offer a promising means of understanding the... more Comparative studies of structure related to function offer a promising means of understanding the significance of differences in cytoarchitecture. Mitochondrial crista structure is linked tightly to mitochondrial function. Non-foveal cone photoreceptors of primates contain considerably more inner segment mitochondria and have higher oxidative enzyme activity than do rods. In addition, it is suggested that light-adapted cones have a higher aerobic ATP demand than light-adapted rods. Therefore, we investigated the oxidative metabolism and three-dimensional membrane architecture of mouse rod and cone inner segment mitochondria. We determined the number, size, cytochrome c oxidase (CO) reactivity, and membrane architecture of rod and middle wavelength-sensitive (M) cone inner segment mitochondria from 21 day old light-adapted C57BL/6 mice using conventional electron microscopy and the three-dimensional approach of single- and double-tilt electron microscope tomography. Fourteen differen...
Abnormal structure and function of astrocytes have been observed within the lamina cribrosa regio... more Abnormal structure and function of astrocytes have been observed within the lamina cribrosa region of the optic nerve head (ONH) in glaucomatous neurodegeneration. Glutamate excitotoxicity-mediated mitochondrial alteration has been implicated in experimental glaucoma. However, the relationships among glutamate excitotoxicity, mitochondrial alteration and ONH astrocytes in the pathogenesis of glaucoma remain unknown. We found that functional N-methyl-d-aspartate (NMDA) receptors (NRs) are present in human ONH astrocytes and that glaucomatous human ONH astrocytes have increased expression levels of NRs and the glutamate aspartate transporter. Glaucomatous human ONH astrocytes exhibit mitochondrial fission that is linked to increased expression of dynamin-related protein 1 and its phosphorylation at Serine 616. In BAC ALDH1L1 eGFP or Thy1-CFP transgenic mice, NMDA treatment induced axon loss as well as hypertrophic morphology and mitochondrial fission in astrocytes of the glial lamina....
Uploads
Papers by Guy Perkins