| Peroxisomes are one of the latest discovered and the least understood of the classical organelles. First described some 50 years ago, these ubiquitous and pleomorphic organelles remain an enigma in terms of their biogenesis, maintenance and degradation. A comprehensive understanding of these mechanisms is critical to understanding the role of these essential organelles in maintaining cellular homeostasis and viability, and ultimately, its role in various human diseases.
Peroxisomes are essential organelles that are required for the cellular metabolism of fatty acids, amino acids, and cellular toxins such as hydrogen peroxides. They also play a crucial role in the biosynthesis of bile salts (salts necessary for lipid digestion), and plasmalogen (essential lipids for the brain, heart and muscles). The importance of peroxisomes for proper cellular function is seen in the numerous inheritable genetic disorders resulting from a mutation in a single enzyme in peroxisomes, or mutation in proteins involved in the assembly of peroxisomes. Although the effects of these mutations in peroxisomal functions/assembly can be seen in various organs, such as, the liver, kidneys, blood and the heart; all peroxisomal disorders affect the development and function of the brain, thus, suggesting the importance of peroxisomes in brain development.
The primary objective of our group is to understand the basic mechanisms involved in the maintenance of peroxisomes in the mammalian cell, particularly in brain development. To achieve this goal, we are focusing on understanding the
1) biogenesis and 2) degradation of peroxisomes using cutting-edge live-cell microscopy techniques on state of the art microscopes in combination with biochemical approaches.
Peroxisome Biogenesis
The long-standing view has been that peroxisomes are autonomous organelles, like mitochondria and chloroplasts that multiply strictly by growth and division. This position is supported by evidence showing that most peroxisomal proteins are synthesized on free ribosomes and are imported directly into peroxisomes from the cytoplasm. However, unlike mitochondria, peroxisomes can disappear from a cell and then be regenerated de novo . This regenerative capacity has led to an alternative view in which other organelles, such as, ER-, participates in the formation and maintenance
of peroxisomal membranes.
Our work with peroxisomes addresses whether the ER plays a role in peroxisomal biogenesis in mammalian cells, and if so, how this is regulated. Towards this end, we have employed diverse live cell fluorescent labeling strategies, including photoactivation, to pulse-label peroxisomal components (including the early event peroxin, PEX16) and to follow their targeting to peroxisomes. Evidence favouring an ER origin of peroxisomal membranes came from our finding that when the ER pool of PEX16-PAGFP was photoactivated and followed over time, the photoactivated molecules redistributed to peroxisomes. This result has helped solidify the view that peroxisomes are derived from the ER and have provided insight into peroxisomes proliferation and maintenance within mammalian cells. Ongoing work in the lab is aimed at using the new live-cell imaging and super-resolution light microscopy strategies to investigate the mechanism of peroxisome formation from the ER.
Figure 1: Cartoon diagram of the de novo biogenesis of peroxisomes. Peroxisomes are formed de novo from the ER by a four step process.
1) PEX16 is directly recruited to the ER where it initates peroxisome biogenesis.
2) PEX3, a membrane anchor protein is recruited to the ER by PEX16.
3) Immature Peroxisomes emerges from the ER. 4) First membrane proteins followed by matrix proteins are recruited to form mature peroxisomes. The mechanism(s) involved in the emergence of immature peroxisomes from the ER remains elusive.
Peroxisome Degradation:
Dysfunctional peroxisomes or excess peroxisomes are recycled through a process called Pexophagy. Pexophagy is the substrate-specific degradation by the autophagy pathways. This process involves the sequestration of peroxisomes by a double membrane structure and delivers peroxisomes to lysosomes for degradation. We believe that either an inhibition of pexophagy or an over-activation of pexophagy can lead to neurodegeneration. To test this hypothesis, a better understanding of pexophagy is required.
To this end, we have used both biochemical and imaging techniques to identify the molecular signal to designate a peroxisome for degradation. Using these various methods, we have shown that the modification of peroxisomal membrane protein with ubiquitin (ubiquitination) is the signal to target peroxisomes to nascent autophagosomes. We are now using advance siRNA gene knockdown techniques in combination with high-throughput screening and fluorescent microscopy to identify all the factors involved in the ubiquitination and targeting of peroxisomes for degradation.
Ultimately, these advances will aid in the future understanding of organelle maintenance and cellular homeostasis, which are essential in understanding all cellular functions and processes. The research into peroxisome biology will not only advance our knowledge of these vital yet understudied organelles, but will also provide a deeper understanding of the role of peroxisomes in the development of the brain and in neurodegenerative diseases.
Figure 2: Mechanism of Pexophagy. A process called Pexophagy, which involves the targeting of peroxisomes to the autophagic pathway, degrades peroxisomes. Peroxisomes are designated for degradation by the accumulation of ubiquitinated membrane proteins. The ubiquitin on peroxisomes recruits ubiquitin binding proteins such as p62 and NBR1 that targets peroxisomes to nascent autophagosomes for degradation. The mechanism of peroxisome ubiquitination is not known.
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Figure1: COS7 cells expressing a ER marker (Red) and a Peroxisome marker (Green)
Fluorescent Loss in Photobleach assay (FLIP) reveals that PEX16 is found in both ER and peroxisomes- The red box indicates the photobleach window (double-click
to play movie)
Movie: Peroxisome with ER. Time laps imaging of peroxisomes (green) and ER (red). Note how peroxisomes move along the ER (double-click to play movie)
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Kim P.K ., Hailey D.W., Mullen R.T., Lippincott-Schwartz J. Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes. (2008) PNAS . 105 (52): 20567-74.
Henderson M.P., Billen L.P., Kim P.K. , Andrews D.W. Cell-free analysis of tail-anchor protein targeting to membranes. (2007) Methods . 41 (4): 427-38.
Kim P.K. , Mullen R.T., Schemann U., Lippincott-Schwartz J. The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ER. (2006) J.Cell Biol . 173 (4): 521-32 .
Brocard C.B., Jedeszko C., Boucher K.K., Kim P.K. , and Walton P.A. Requirement for microtubules and dynein-motors in the earliest stages of peroxisome biogenesis. (2005) Traffic 6 (5): 386-95.
Kim P.K. , Annis M.G., Dlugosz P.J., Leber B., Andrews, D.W. During apoptosis Bcl-2 changes membrane topology at both the endoplasmic reticulum and mitochondria. (2004) Molecular Cell . 14 (4): 523-9.
McCartney A.W., Dyer J.M., Dhanoa P.K., Kim P.K. , Andrews D.W., McNew J.A., Mullen R.T. Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini. (2004) Plant J . 37 (2): 156-73.
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