Biomedical subjects

Paul A Walton

Publications and source records attributed to Paul A Walton.

7 recordsLinked to original sources

PubMed2006

Peroxisomes and aging.

Peroxisomes are indispensable for proper functioning of human cells. They efficiently compartmentalize enzymes responsible for a number of metabolic processes, including the absolutely essential beta-oxidation of specific fatty acid chains. These and other oxidative reactions produce hydrogen peroxide, which is, in most instances, immediately processed in situ to water and oxygen. The responsible peroxidase is the heme-containing tetrameric enzyme, catalase. What has emerged in recent years is that there are circumstances in which the tightly regulated balance of hydrogen peroxide producing and degrading activities in peroxisomes is upset-leading to the net production and accumulation of hydrogen peroxide and downstream reactive oxygen species. The factor most essentially involved is catalase, which is missorted in aging, missing or present at reduced levels in certain disease states, and inactivated in response to exposure to specific xenobiotics. The overall goal of this review is to summarize the molecular events associated with the development and advancement of peroxisomal hypocatalasemia and to describe its effects on cells. In addition, results of recent efforts to increase levels of peroxisomal catalase and restore oxidative balance in cells will be discussed.

Acatalasia↗

PubMed2006

Galpha13 activation rescues moesin-depletion induced apoptosis in F9 teratocarcinoma cells.

Mouse F9 cells differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm in response to RA and dibutyryl (db-) cAMP. G protein signaling either blocks or mimics RA-induced differentiation, the latter signaling through the Wnt-beta-catenin pathway. In our study, we found that a constitutively active Galpha13 mutant induces F9 cells to differentiate into parietal endoderm in the absence of exogenous agents. Galpha13 expression and subsequent differentiation are accompanied by beta-catenin translocation to the nucleus. Differentiation and changes in cell morphology are supported by rearrangements to the F-actin cytoskeleton. ERM (ezrin-radixin-moesin) proteins, known to link F-actin to transmembrane receptors, are also redistributed during differentiation. Furthermore, morpholino antisense and shRNA approaches show that moesin expression is essential since its knockdown leads to altered F-actin distribution and subsequent apoptosis. Moesin-depleted cells, however, remain attached to the substrate when Galpha13 is constitutively expressed, but they do not differentiate into extraembryonic endoderm. Our study demonstrates a link between Galpha13 signaling that regulates differentiation of F9 cells through primitive to parietal endoderm and a moesin requirement for cell survival.

Actins↗

PubMed2006

Hypocatalasemic fibroblasts accumulate hydrogen peroxide and display age-associated pathologies.

Human epidemiological studies point to an association of hypocatalasemia and an increased risk of age-related disease. Unfortunately, the cellular and molecular manifestations of hypocatalasemia are only poorly understood. In this analysis, we have extensively characterized hypocatalasemic human fibroblasts and report that they amass hydrogen peroxide and are oxidatively damaged. Protein and DNA alike are affected, as are functioning and biogenesis of peroxisomes - the subcellular organelles which normally house catalase. Despite these pathologies and their relative inability to grow, the cells do not appear to be intrinsically senescent. With the goal of restoring oxidative balance and perhaps reversing some of the accumulated damage to critical cellular components, we transduced hypocatalasemic fibroblasts with a form of catalase specifically designed to efficiently traffic to peroxisomes. We show the strategy is extremely effective, with dramatic reductions seen in cellular hydrogen peroxide levels. Future longitudinal studies aimed at examining the effects of a more continuous and long-term protein therapy may now commence.

Age Factors↗

PubMed2005

Requirement for microtubules and dynein motors in the earliest stages of peroxisome biogenesis.

Our aim was to determine the role of microtubules in the biogenesis of peroxisomes. Fusion experiments between human PEX16- and PEX1-mutant cells in the presence of nocodazol implied that microtubules were not required for import of proteins into the peroxisomal matrix after cell fusion complementation. We further studied the importance of microtubules in the early stages of peroxisome biogenesis following the microinjection complementation of PEX16-mutant cells. In the absence of nocodazol, nuclear microinjection of plasmids expressing EGFP-SKL and Pex16p in PEX16-mutant cells resulted in the accumulation of EGFP-SKL into newly formed peroxisomes. However, pretreatment of the cells with nocodazol, prior to microinjection, resulted in the inhibition of complementation of the PEX16 mutant and the cytosolic location of the EGFP-SKL. In addition, coexpression of a dominant-negative CC1 subunit of the dynein/dynactin motor complex resulted in the inability to complement PEX16-mutant cells. Both of these treatments resulted in the cytosolic localization of expressed Pex16p. Our results demonstrate that the formation of peroxisomes via the preperoxisomal compartment is dependent upon microtubules and minus-end-directed motor proteins and that the inhibition described above occurs at a step that precedes the association of Pex16p with the vesicles that would otherwise become the peroxisomes.

Cell Fusion↗

PubMed2004

The Pleckstrin homology domain of CK2 interacting protein-1 is required for interactions and recruitment of protein kinase CK2 to the plasma membrane.

CKIP-1 is a recently identified interaction partner of protein kinase CK2 with a number of protein-protein interaction motifs, including an N-terminal pleckstrin homology domain. To test the hypothesis that CKIP-1 has a role in targeting CK2 to specific locations, we examined the effects of CKIP-1 on the localization of CK2. These studies demonstrated that CKIP-1 can recruit CK2 to the plasma membrane. Furthermore, the pleckstrin homology domain of CKIP-1 was found to be required for interactions with CK2 and for the recruitment of CK2 to the plasma membrane. In this regard, point mutations in this domain abolish membrane localization and compromise interactions with CK2. In addition, replacement of the pleckstrin homology domain with a myristoylation signal was insufficient to elicit any interaction with CK2. An investigation of the lipid binding of CKIP-1 reveals that it has broad specificity. A comparison with other pleckstrin homology domains revealed that the pleckstrin homology domain of CKIP-1 is distinct from other defined classes of pleckstrin homology domains. Finally, examination of CK2alpha for a region that mediates interactions with CKIP-1 revealed a putative HIKE domain, a complex motif found exclusively in proteins that bind pleckstrin homology domains. However, mutations within this motif were not able to abolish CKIP-1-CK2 interactions suggesting that this motif by itself may not be sufficient to mediate interactions. Overall, these results provide novel insights into how CK2, a predominantly nuclear enzyme, is targeted to the plasma membrane, and perhaps more importantly how it may be regulated.

Amino Acid Motifs↗

PubMed2003

Protein structure and import into the peroxisomal matrix.

Proteins destined for the peroxisomal matrix are synthesized in the cytosol, and imported post-translationally. It has been previously demonstrated that stably folded proteins are substrates for peroxisomal import. Mammalian peroxisomes do not contain endogenous chaperone molecules. Therefore, it is possible that proteins are required to fold into their stable, tertiary conformation in order to be imported into the peroxisome. These investigations were undertaken to determine whether proteins rendered incapable of folding were also substrates for import into peroxisomes. Reduction of albumin resulted in a less compact tertiary structure as measured by analytical centrifugation. Microinjection of unfolded albumin molecules bearing the PTS1 targeting signal resulted in their import into peroxisomes. Kinetic analysis indicated that native and unfolded molecules were imported into peroxisomes at comparable rates. While import was unaffected by treatment with cycloheximide, hsc70 molecules were observed to be imported along with the unfolded albumin molecules. These results indicate that proteins, which are incapable of assuming their native conformation, are substrates for peroxisomal import. When combined with previous observations demonstrating the import of stably folded proteins, these results support the model that tertiary structure has no effect on protein import into the peroxisomal matrix.

Cycloheximide↗

PubMed2002

Peroxisome senescence in human fibroblasts.

The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this report, we characterize age-related changes in peroxisomes of human cells. We show that aging compromises peroxisomal targeting signal 1 (PTS1) protein import, affecting in particular the critical antioxidant enzyme catalase. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite hydrogen peroxide, and we present evidence that this increased load of reactive oxygen species may further reduce peroxisomal protein import and exacerbate the effects of aging.

Aging↗