# Lecture 15: The Peroxisome

## Cell Biology

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## Learning Objectives

By the end of this lecture, students will be able to:

1. Describe the structure and major metabolic functions of peroxisomes
2. Explain the role of peroxisomes in fatty acid beta-oxidation and ROS metabolism
3. Describe the peroxisomal protein import machinery (PTS1/PTS2 pathways)
4. Explain peroxisome biogenesis and its relationship to the ER
5. Discuss peroxisomal disorders (Zellweger syndrome, X-linked adrenoleukodystrophy)

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## Lecture Content

### I. Peroxisome Structure and Overview

Peroxisomes are single-membrane-bound organelles ranging from 0.1 to 1 micrometer in diameter, found in virtually all eukaryotic cells. Their abundance varies dramatically by cell type, from 100 to 1,000 in hepatocytes to relatively few in most other cell types. They were originally identified by Christian de Duve (Nobel Prize, 1974) and named for their content of hydrogen peroxide-producing oxidases and the H2O2-decomposing enzyme catalase. Peroxisomes have a dense granular matrix, and in some species, they contain a crystalline core of urate oxidase.

A critical distinguishing feature of peroxisomes is that they contain no DNA or ribosomes. All peroxisomal proteins are encoded by nuclear genes and imported from the cytoplasm. Furthermore, unlike mitochondria, peroxisomes import their matrix proteins in a **fully folded state**, and can even import assembled oligomeric complexes.

### II. Metabolic Functions of Peroxisomes

Peroxisomes carry out several essential metabolic functions. **Hydrogen peroxide metabolism** is central to their identity. Multiple oxidases within the peroxisome generate H2O2 as a byproduct of their reactions, including fatty acyl-CoA oxidase, D-amino acid oxidase, and urate oxidase. **Catalase**, the most abundant peroxisomal enzyme, decomposes this potentially toxic H2O2 in two ways: a catalatic reaction (2H2O2 -> 2H2O + O2) and peroxidative reactions (H2O2 + RH2 -> R + 2H2O), the latter being important for detoxification reactions such as ethanol oxidation in hepatocytes.

**Fatty acid beta-oxidation** in peroxisomes specifically handles very long chain fatty acids (VLCFAs, with chains longer than 22 carbons). Peroxisomes shorten VLCFAs to medium-chain products, which are then transferred to mitochondria for complete oxidation. Peroxisomal beta-oxidation differs from mitochondrial beta-oxidation in important ways: the first step uses an FAD-linked acyl-CoA oxidase that produces H2O2 (rather than feeding electrons to the ETC via FADH2), and the process does not directly generate ATP. Peroxisomes also oxidize branched-chain fatty acids (phytanic acid, pristanic acid), dicarboxylic acids, and bile acid intermediates (DHCA, THCA).

**Plasmalogen synthesis** is another essential peroxisomal function. Plasmalogens are ether phospholipids with a vinyl ether bond at the sn-1 position, abundant in brain myelin, heart, and immune cells. The first two enzymatic steps of plasmalogen synthesis, catalyzed by DHAP acyltransferase and alkyl-DHAP synthase, occur in peroxisomes. Deficiency of plasmalogen synthesis causes severe neurological defects.

Peroxisomes also participate in bile acid synthesis (side-chain oxidation of cholesterol intermediates), glyoxylate metabolism (converting glyoxylate to glycine, with deficiency causing primary hyperoxaluria type 1), alpha-oxidation of branched-chain fatty acids such as phytanic acid (deficiency causes Refsum disease), amino acid catabolism, and reactive oxygen species scavenging through enzymes including superoxide dismutase and glutathione peroxidase.

<image>Overview of peroxisomal metabolic functions. Panel A: Central diagram of a peroxisome with key metabolic pathways labeled — (1) Very long chain fatty acid beta-oxidation (C26 -> C8 product transferred to mitochondrion), (2) H2O2 production by oxidases and decomposition by catalase, (3) Plasmalogen synthesis (first two steps), (4) Bile acid synthesis. Panel B: Comparison of beta-oxidation in peroxisomes vs. mitochondria — highlighting that the first step in peroxisomes uses acyl-CoA oxidase producing H2O2 (decomposed by catalase), whereas mitochondria use acyl-CoA dehydrogenase feeding electrons to the ETC via FADH2.</image>

### III. Peroxisomal Protein Import

All approximately 100 peroxisomal matrix proteins and peroxisomal membrane proteins are nuclear-encoded and imported from the cytoplasm. Uniquely among organelles, matrix proteins are imported in a **fully folded state** and can even enter as oligomeric complexes through "piggyback" import.

Two **peroxisomal targeting signals** direct proteins to the peroxisomal matrix. **PTS1** is a C-terminal tripeptide (SKL or conservative variants) used by most peroxisomal matrix proteins and recognized by the cytoplasmic receptor **Pex5**. **PTS2** is an N-terminal nonapeptide used by fewer proteins (such as thiolase and phytanoyl-CoA hydroxylase), recognized by the receptor **Pex7**, and cleaved after import in mammals.

The import mechanism proceeds through a series of coordinated steps. Pex5 (or Pex7) binds the cargo protein in the cytoplasm. The receptor-cargo complex docks at the peroxisomal membrane through the docking complex (Pex13 and Pex14). The cargo is translocated across the membrane through a transient pore, likely formed by Pex5 itself or in conjunction with Pex14. After cargo release into the matrix, Pex5 is mono-ubiquitinated and recycled to the cytoplasm, a process powered by the AAA-ATPases Pex1 and Pex6. As a quality control measure, polyubiquitinated Pex5 is degraded by the proteasome. Peroxisomal membrane proteins are inserted through a separate pathway involving Pex19 (a cytoplasmic chaperone and receptor) and Pex3 (a membrane receptor and insertase), and some may traffic through the ER before reaching peroxisomes.

### IV. Peroxisome Biogenesis

Peroxisomes can form through two complementary pathways. In **growth and division**, pre-existing peroxisomes grow by importing lipids and proteins and then divide by fission. Pex11 family proteins elongate the peroxisome, and fission is carried out by Drp1 and Fis1, the same machinery used for mitochondrial fission. In **de novo formation from the ER**, peroxisomal membrane proteins such as Pex3 and Pex16 are first inserted into the ER membrane, and pre-peroxisomal vesicles bud from the ER and mature into functional peroxisomes. Evidence for this pathway comes from the observation that cells completely lacking peroxisomes can regenerate them from the ER when the missing peroxin gene is re-expressed. The current consensus is that both pathways operate: growth and division for routine maintenance, and the ER-derived pathway for de novo biogenesis. Because peroxisomes cannot synthesize their own membrane lipids, they receive lipids from the ER through vesicular transport or via lipid transfer proteins at ER-peroxisome contact sites.

### V. Peroxisomal Disorders

**Peroxisome biogenesis disorders (PBDs)** result from mutations in peroxin (PEX) genes that disrupt peroxisome formation or function. The **Zellweger spectrum disorders** range from the most severe form, **Zellweger syndrome** (cerebro-hepato-renal syndrome), through intermediate **neonatal adrenoleukodystrophy (NALD)**, to the mildest form, **infantile Refsum disease (IRD)**. Zellweger syndrome involves complete loss of peroxisome function due to mutations in PEX1 (the most common), PEX5, PEX6, PEX12, PEX26, or other PEX genes. Clinical features include severe hypotonia, seizures, hepatomegaly, renal cysts, and a characteristic facial appearance. Laboratory findings show elevated VLCFAs and phytanic acid in blood and reduced plasmalogens. The disease is fatal in the first year of life. **Rhizomelic chondrodysplasia punctata (RCDP)** results from defects in PEX7 (the PTS2 receptor), DHAP acyltransferase, or alkyl-DHAP synthase, leading to impaired plasmalogen synthesis and causing limb shortening, cataracts, and intellectual disability.

Among **single enzyme deficiencies**, **X-linked adrenoleukodystrophy (X-ALD)** is the most common peroxisomal disorder, affecting approximately 1 in 17,000 males. It is caused by mutations in the ABCD1 gene, which encodes ALDP, a peroxisomal ABC transporter that imports VLCFA-CoA into the peroxisome for beta-oxidation. Without functional ALDP, VLCFAs accumulate. The disease manifests in several clinical forms: childhood cerebral ALD features progressive inflammatory demyelination, adrenomyeloneuropathy (AMN) is an adult-onset form with spinal cord and peripheral neuropathy, and adrenal insufficiency can also occur. This disease was featured in the film "Lorenzo's Oil." Hematopoietic stem cell transplant can be curative if performed early, and gene therapy trials are showing promise. **Refsum disease** results from defective phytanoyl-CoA hydroxylase, causing phytanic acid accumulation and producing retinitis pigmentosa, peripheral neuropathy, and cerebellar ataxia, treatable by dietary restriction of phytanic acid. **Primary hyperoxaluria type 1** is caused by deficient alanine:glyoxylate aminotransferase (AGT), leading to oxalate overproduction, calcium oxalate kidney stones, and eventual renal failure.

<image>Peroxisome biogenesis and import pathway. Panel A: Peroxisomal protein import — PTS1 cargo protein bound by Pex5 receptor in the cytoplasm; complex docks at peroxisomal membrane via Pex13/Pex14; cargo translocated in folded state through a transient pore; Pex5 recycled via mono-ubiquitination and Pex1/Pex6 AAA-ATPases. PTS2 pathway shown in parallel with Pex7 receptor. Panel B: Peroxisome biogenesis — dual pathways: (1) growth and division of existing peroxisomes (elongation by Pex11, fission by Drp1/Fis1), and (2) de novo formation from the ER (Pex3/Pex16 insertion into ER, pre-peroxisomal vesicle budding, maturation into functional peroxisome).</image>

### VI. Peroxisome-Organelle Interactions

Peroxisomes do not function in isolation but maintain extensive interactions with other organelles. **Peroxisome-mitochondria cooperation** is particularly important: peroxisomes shorten VLCFAs and transfer the products to mitochondria for complete oxidation, both organelles contribute to ROS metabolism, they share the fission machinery (Drp1 and Fis1), and metabolites such as acetyl-CoA and NAD+/NADH are channeled between them.

**Peroxisome-ER interactions** are mediated by contact sites involving VAP proteins on the ER and ACBD5 on the peroxisome. The ER supplies membrane lipids to peroxisomes, and plasmalogen synthesis requires enzymes in both compartments. Cholesterol and bile acid synthesis intermediates also shuttle between the two organelles. **Peroxisome-lipid droplet interactions** facilitate fatty acid trafficking, and physical contacts between the two organelles have been observed by microscopy.

**Pexophagy**, the selective autophagy of peroxisomes, removes excess or damaged peroxisomes. Ubiquitination of peroxisomal membrane proteins triggers pexophagy, with NBR1 and p62 serving as the autophagy receptors that link the ubiquitinated peroxisome to the autophagosome.

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