Tuesday, October 12, 2010

New insights into peroxisome function and an in vivo peroxisomal trafficking assay

Peroxisomal pipecolate pathway involved in L-lysine catabolism in brain
Therapeutic modulation of cerebral L-lysine metabolism in a mouse model for glutaric aciduria type I.
Sauer et al
Department of General Paediatrics,
Division of Inborn Metabolic Diseases, University Children’s Hospital, Hiedelberg.
Published in journal Brain (A journal of neurology) published online: October 4, 2010
Impact factor of journal BRAIN - 9.49

L-lysine catabolism proceeds via mitochondrial saccharopine pathway or peroxisomal pipecolate pathway. Human L-pipecolate oxidase(PIPOX) is a peroxisomal enzyme carrying C-terminal PTS1 motif (KAHL). Peroxisome Biogenesis Disorder patients lacking peroxisomes or defective in matrix protein import, lack L-pipecolate oxidase activity. Type I Glutaric aciduria patients have genetic defect in glutaryl-CoA dehydrogenase, which leads to accumulation of neurotoxic metabolites. In this paper, Authors used animal model of this disease(gcdh deficient mice) to study the effects of dietary interventions and underlying mechanism of their therapeutic effect. They observed that in brain, Lysine catabolism occurs mainly by peroxisomal L-pipecolate pathway whereas in hepatic cells, it occurs via mitochondrial pathway.

This paper demonstrates that peroxisomes play an important role in brain, by preventing the accumulation of toxic metabolites in L-lysine catabolism. Clofibrate treatment in these mice could further lower the glutaric acid accumulation, which shows that peroxisomal pipecolate pathway could be targeted to recover the defects in mitochondrial enzyme glutaryl-CoA dehydrogenase. Why brain cells prefer action of PIPOX for lysine degradation over saccharopine pathway remains to be better understood.

Peroxisomes as a tool to study motor proteins in cellular traffic
Probing intracellular motor protein activity using an inducible cargo trafficking assay
Kapitein et al.
From the group of Prof. Casper Hoogenraad, Erasmus MC, Rotterdam
Published in Biophysical Journal (Cell Press). Impact Factor - 4.39

Various motor proteins mediate cellular movements on the cytoskeleton network. Kinesins and dyneins are generally involved in long range transport over microtubule network while myosin motors mediate short range movement over actin network. Though the knowledge about how individual motor proteins work was deduced from in vitro studies. In vivo functional dynamics of the motor proteins is still elusive owing to diversity and complexity of the various motor proteins involved in single event. Kapitein et al. report in this paper, an inducible cargo trafficking assay which can be employed in live cells with great control. The assay is based on peroxisomes, so called in vivo peroxisomal trafficking assay. The assay is based on the dimerisation property of two proteins FKBP and FRB on addition of cell permeable rapalog. Rapalogs are non-immunosuppressive analogs of rapamycin which can be used conveniently in ARGENT regulated Heterodimerization Kit produced by Ariad Pharmaceuticals. Pex3 is fused to RFP(for visualisation) and FKBP, whereas the motor protein to be studied is fused to FRB. Both constructs are coexpressed in mammalian cells (in this paper, COS7 cells). Upon addition of rapalog, heterodimeraisation is induced leading to recruitment of desired motor protein to peroxisomes and initiated movement of peroxisomes. Since peroxisomes in COS7 cells are relatively immobile and clustered around nucleus. Authors observed that Kinesins induce +end directed movement of peroxisomes leading to peripheral distribution of peroxisomes. Dyneins result in -end directed movement of peroxisomes towards nucleus. As expected, myosins induce only short range movement.

Authors also did simultaneous visualization of peroxisomes and microtubules and tracked the movements. Peroxisomes where labeled with GFP using Pex26 targeting information, while microtubules were labeled with mcherry. They observed that peroxisomes often pause at microtubule intersections and resume their movement on same or another microtubule after the second microtubule gets depolymerised till the junction.

This in vivo peroxisomal trafficking assay was used by authors in another paper (Kapitein et al, Current Biology, 2010) to demonstrate that kinesins are preferred for trafficking in axons and dyneins are preferred in dendrites in hippocampal neurons. This demonstrates the this assay can be utilized to study individual motor proteins as well as combination of them.

Though this study and the assay were not designed to study peroxisome movement, but to study the properties of motor proteins. But since our knowledge about endogenous movement of peroxisomes and the motor proteins involved is scanty, so I believe that this assay can be directed for peroxisome research also.