Redox Regulation of Homeostasis and Proteostasis in Peroxisomes

Abstract

Peroxisomes are highly dynamic intracellular organelles involved in a variety of metabolic functions essential for the metabolism of long-chain fatty acids, d-amino acids, and many polyamines. A byproduct of peroxisomal metabolism is the generation, and subsequent detoxification, of reactive oxygen and nitrogen species, particularly hydrogen peroxide (H₂O₂). Because of its relatively low reactivity (as a mild oxidant), H₂O₂ has a comparatively long intracellular half-life and a high diffusion rate, all of which makes H₂O₂ an efficient signaling molecule. Peroxisomes also have intricate connections to mitochondria, and both organelles appear to play important roles in regulating redox signaling pathways. Peroxisomal proteins are also subject to oxidative modification and inactivation by the reactive oxygen and nitrogen species they generate, but the peroxisomal LonP2 protease can selectively remove such oxidatively damaged proteins, thus prolonging the useful lifespan of the organelle. Peroxisomal homeostasis must adapt to the metabolic state of the cell, by a combination of peroxisome proliferation, the removal of excess or badly damaged organelles by autophagy (pexophagy), as well as by processes of peroxisome inheritance and motility. More recently the tumor suppressors ataxia telangiectasia mutate (ATM) and tuberous sclerosis complex (TSC), which regulate mTORC1 signaling, have been found to regulate pexophagy in response to variable levels of certain reactive oxygen and nitrogen species. It is now clear that any significant loss of peroxisome homeostasis can have devastating physiological consequences. Peroxisome dysregulation has been implicated in several metabolic diseases, and increasing evidence highlights the important role of diminished peroxisomal functions in aging processes.

FIGURE 1.

Metabolic functions of peroxisomes in different organisms. Oxidation of fatty acids and H₂O₂ degradation by catalase is a common function that is performed by all peroxisomes irrespective of the organism type. Other functions are detailed in plants, protozoa, fungi, and animals.

FIGURE 2.

Quality control checks for peroxisomal proteins. Peroxisome-destined proteins have various checks, before and after import, to ensure proper protein folding. In mammalian cells, the first check relies on newly synthesized proteins from ribosomes containing the correct sequence to ensure that proper configuration is achieved. This is further assisted during the second quality control check, with correct folding assisted by the heat shock protein 70 (HSP70). If the newly synthesized protein is incorrectly folded, it is targeted for degradation by the 26S Proteasome while still in the cytoplasm. The third check relies on the presence of the correct peroxisome targeting signal (PTS). Without the correct PTS signal, PEX5 is unable to recognize the peroxisome-targeted protein, resulting in degradation. Upon binding to PEX5, fully folded peroxisome proteins are imported into the peroxisome. The fourth check relies on the LonP2 enzyme, which can act as either a chaperone or a protease. Due to the highly oxidizing environment, peroxisomal proteins are prone to either lose their configuration or become oxidized (or both). If the protein loses proper folding, LonP2 can assist in helping to refold the protein. However, if the protein becomes oxidized, LonP2 will degrade the damaged protein.

FIGURE 3.

Import of peroxisomal proteins. Newly synthesized peroxisome-targeted proteins are directed to the peroxisome predominantly due to recognition of the peroxisome targeting signal 1 (C’-PTS1), a tripeptide, located on the carboxy terminal of peroxisome-targeted proteins. In yeast, to ensure correct protein folding, various heat shock proteins, such as the heat shock 70 (HSP70) chaperone protein, bind to the newly synthesized peptide and assist in folding. The newly folded protein is recognized in the cytosol by the soluble receptor Pex5. In turn, Pex5 binds to the protein and brings the protein into the peroxisome, where it docks with Pex14, before being recycled back to the cytosol. A small number of peroxisomal proteins contain a conserved peptide recognition sequence, peroxisome targeting signal 2 (PTS2-N’), including catalase, located on the amino terminus. Again, various heat shock chaperones bind to the newly synthesized protein with evidence indicating the interaction of Hsc70 and HSP40 with PTS2-containing peptide, to ensure proper folding. Pex7, which recognizes the PTS2-containing sequence, along with the cofactors Pex18 and Pex21, brings the protein to the peroxisome, where it interacts with the docking complex, comprised of Pex13 and Pex14. Upon import, the PTS2 sequence is cleaved off.

FIGURE 4.

Peroxisome biogenesis. Details of peroxisome generation by de novo synthesis, and by growth and fission, are shown here. In the de novo pathway, peroxisomes are formed via budding from the endoplasmic reticulum (ER) and pairwise heterotypic fusion of two vesicles. Initiation begins by the peroxisomal membrane proteins (PMPs) entering into the ER via Sec61 or GET3 and traffic through the ER to get into the vesicles. One of the vesicles contains a set of RING finger proteins (PEX2, PEX10, and PEX12), while the other vesicle contains a set of docking group proteins. Following heterotypic fusion, both vesicles merge and form an import complex group. PEX1 and PEX6 are found on separate preperoxisomal vesicles and are necessary for heterotypic fusion. Once peroxisomes are formed, they grow by import of matrix and membrane-targeted proteins via the import receptor PEX5/PEX7 and PEX19, respectively, to form mature peroxisomes. In turn, mature peroxisomes grow by fission which begins with membrane remodeling and elongation by PEX11. The elongated extension grows and dynamin-related proteins (DRPs), which are also involved in fission of mitochondria, including the Fis1 (or DLP1; dynamin-like proteins in mammalian cells) are recruited from the cytosol to help with the fission and formation of new peroxisomes.

FIGURE 5.

Three mechanisms of pexophagy in mammalian cells. Three different forms of ubiquitin (Ub)-dependent pexophagy have been shown in mammals. 1) First, the ubiquitinylation of the overexpressed peroxisomal membrane-associated protein, PEX3, serves as a signal for the adaptor complex NBR1/p62, causing an induction of pexophagy. 2) The second method of pexophagy is triggered by high amounts of reactive oxygen and nitrogen species (ROS, RNS), which cause the activation of the ATM-TSC2 signaling node, which resides on the peroxisome membrane. ATM performs two functions: a) activation of the AMPK-TSC2 signaling node and the suppression of the mTORC1, together inducing autophagy via the activation of ULK1. b) ATM phosphorylates PEX5 at S141 position, which serves as a signal for the Ub E3 ligase (PEX2, PEX10, and PEX12) and the ubiquitinylation at K209. Following ubiquitinylation, the autophagic receptor/adaptor p62 is activated, resulting in the induction of pexophagy by binding the targeted peroxisome to a phagophore. 3) The third pathway begins by the mono-ubiquitinylation of the peroxisome matrix protein import receptor, PEX5, on Cys-11 and blocks the recycling of this mono-ubiquitinylated PEX5 (due to the failure of the cargo delivery into the matrix of the peroxisome) from the peroxisomal cytosol via the receptor recycling complex (AAA-ATPases, PEX1, and PEX6 anchored at peroxisome membrane via PEX26), which serves as a signal for pexophagy.

FIGURE 6.

Domains and motifs of the pexophagy receptors in yeast and mammals. In Saccharomyces cerevisiae, Atg36, and Pichia pastoris, Atg30, serve as pexophagy receptors. They contain a potential globular domain (GlobDom) as defined by Russell and Linding. Both Atg36 and Atg30 directly recognize proteins on the peroxisome surface and interact with the autophagy machinery via their LIR motifs and Atg11 binding sites. The mammalian systems have two pexophagy receptors, p62 and NBR1, also called adaptors. Both receptors/adaptors contain several conserved domains and motifs which include the following: PB1 (Phox/Bem1p) domain at the amino terminus, which is involved in the interaction with another p62 molecule; ZZ-type zinc finger domain in the middle; LIR domain for interacting with autophagic machinery; and the UBA domain at the carboxy terminus, which binds to ubiquitinated cargo. In addition, the NBR1 adaptor contains the CC1 and CC2 domains for homodimerization, the ZZ domain, and an amphipathic JUBA domain involved in lipid binding (allowing for direct peroxisome binding). Additionally, p62 has a KIR motif (Keap 1-interacting) nuclear localization signal (NLS1 and NLS2), nuclear export signal (NES), and TRAF6 binding domain (TBS).

FIGURE 7.

Mechanisms of pexophagy in yeast. In yeast, pexophagy signaling is dependent on mitogen-activated protein kinase (MAPK) pathways (Mid2-Slt2 cascade). It may also be triggered by environmental (external or internal) factors such as signals related to the status of metabolic needs, or to high amount of reactive oxygen species generated by damaged or superfluous peroxisomes. In contrast to ubiquitinylation as observed in mammalian systems, phosphorylation of pexophagy receptors S. cerevisiae Atg36 or P. pastoris Atg30 in yeast serve as signals to induce pexophagy.

FIGURE 8.

Binding of NBR1 and p62 to peroxisomes during pexophagy. During pexophagy, p62 and NBR1 bind with LC3 on the phagophore via the LIR domain and with ubiquitinylated peroxins. Together, these serve as signals for pexophagy via the UBA domain. Additionally, NBR1 has a JUBA domain so it can bind directly to the peroxisome membrane, and it is considered to be an endogenous receptor of pexophagy.

FIGURE 9.

Decline in peroxisome function with age and/or senescence. With age, peroxisome activity declines. Peroxisomes from low-passage cells show efficient fatty acid oxidation (green). However, upon increasing passages, peroxisome efficiency declines (yellow), until peroxisome function is highly inefficient (red). To compensate, peroxisome proliferation occurs in late-passage cells. However, increasing peroxisome number is not enough to compensate for the high amount of damage that has already occurred, especially as many of the additional peroxisomes will also be damaged.

FIGURE 10.

Decline in catalase with age and/or senescence. Catalase is one of the primary peroxisomal enzymes to breakdown hydrogen peroxide. However, catalase contains a noncanonical peroxisomal-targeting signal, that decreases its recognition affinity by Pex5, compared with the stronger recognition elicited by the traditional signal. The decreased catalase targeting to the peroxisome is further compounded with age. Unlike low-passage cells, senescent cells show a decreased amount of Pex5 present in the cell, combined with further decreased recognition. This in turn lowers the ability of catalase to be targeted to the peroxisome. In addition, high-passage cells show decreased import efficiency by peroxisome import proteins, thus further limiting catalase import. As a result, peroxisomes from senescent cells have decreased function, and the organelle has a higher propensity to cause oxidative damage to itself and to surrounding cellular structures.