Peroxisome Functions and Biogenesis

Peroxisome Functions and Biogenesis

• Peroxisomes break down long fatty acid chains, enabling their use in mitochondria for energy production.

• They detoxify hydrogen peroxide (H₂O₂) produced by cell metabolism, converting it to water and oxygen, using catalase.

• Peroxisomes detoxify reactive oxygen species (ROS) via oxidase enzymes, preventing oxidative stress.

• Bile and cholesterol synthesis occur in peroxisomes within the liver.

• They participate in plasmalogen synthesis and photorespiration by recycling intermediates.

• Peroxisomes also play a role in immune and metabolic regulation.

• Peroxisome formation (biogenesis) can be identified through sequence comparisons of peroxisome targeting signals (PTS).

• PTS1 is at the C-terminus and PTS2, at the N-terminus.

• These PTSs are recognized and bound by PEX5 and PEX7 proteins.

• Peroxisomes sprout from the endoplasmic reticulum (ER) and contain precursor proteins.

• Pre-peroxisomes fuse through fission to form immature peroxisomes.

Protein Targeting to Peroxisomes

• Defective peroxisomes can be induced in yeast cultures to study targeting mechanisms.
• Yeast cells are diluted and cultured in aliquots to individually observe cells.
• Diluted cells are plated and grown in different media to observe their growth and behavior in various conditions.
• Mutant cells are then identified based on the growth conditions.

PEX5 Shuttling and Protein Import

• PEX5 proteins bind to proteins with PTS1 signals.
• The complex moves to the peroxisomal membrane.
• PEX5 interacts with PEXs 13 and 14, forming a translocation complex.
• PTS1 is directed into the peroxisome, with PEX5 ubiquitinated and transported out of the organelle.
• PEX5 returns to the cytosol via an ATP-dependent transport mechanism.

Peroxisome Disorders

• X-ALD (X-linked adrenoleukodystrophy): A mutation in the ABCD1 gene affects peroxisome transporters, preventing very long fatty acid chain breakdown. This disrupts plasmalogen synthesis, impacting heart and brain function. Diagnosable through elevated very long-chain fatty acids (VLCFAs) in the blood coupled with the ABCD1 gene mutation.

• ZSD (Zellweger spectrum disorder): Mutations in PEX genes (e.g., PEX1, 6, 12), causing defective peroxisome biogenesis. Results in reduced plasmalogen production, increased VLCFAs, and intermediate bile acid accumulation. An autosomal recessive disorder.

Cholesterol Synthesis and Regulation

• Density Centrifugation separates blood components for cholesterol analysis. Multiple spins separate plasma components, with chylomicrons at the top layer.

• Synthesis: Cholesterol synthesis begins with Acetyl CoA, converting to HMG CoA, further metabolized to mevalonate, which is a key precursor to cholesterol.

• Statins: These drugs lower blood cholesterol by inhibiting the conversion of HMG CoA to mevalonate, reducing intracellular cholesterol and increasing cholesterol receptor activity.

• PCSK9 inhibitors: These inhibitors of PCSK9 protein prevent LDL receptor degradation, extending their life and promoting cholesterol uptake.

• SREBP (Sterol Regulatory Element Binding Protein) is a transcription factor regulating cholesterol in cells.

• It activates HMG-CoA reductase and LDL receptors transcription.

• High cholesterol represses SREBP by keeping it in the ER; low cholesterol activates SREBP for cholesterol synthesis regulation.

Protein Lipidation and Membrane Targeting

• Protein Glypiation: Addition of a glycosylphosphatidylinositol (GPI) anchor (a lipid-sugar combination) as a post-translational modification. This membrane attachment is synthesized in the ER, requiring a signal peptide for binding.

• Protein Cholestorylation: Hedgehog proteins are examples of proteins that experience this modification. This involves cholesterol binding, structural rearrangement of the protein, and finally the covalent attachment to cholesterol itself.

• Protein Isoprenylation: The addition of isoprenoid lipids (farnesyl or geranylgeranyl) to cysteine residues at the C-terminus. The attachment serves as a hydrophobic membrane anchor.

• Protein Acylation: The covalent attachment of fatty acid chains (e.g., myristoylation, palmitoylation) to proteins via amide, thioester, or ester bonds. This modification increases hydrophobicity and facilitates membrane interaction.

Detection Methods for Lipidation

• Palmitate replacement: Radioactively or fluorescently-labeled palmitic acid is added to identify the attachment process.

• Click Chemistry: A specific chemical tag is attached to a fatty acid, enabling complementary reaction detection.

• Bioorthogonal Chemistry: More advanced chemistries allow detection without interfering with other biological molecules.

Cytoskeleton Structures and Functions

• Microtubules: Hollow tubes composed of alpha and beta tubulin dimers. They provide cellular structure and shape, regulated by microtubule-organizing centers (MTOCs). Polymerization (GTP) and depolymerization (GDP) are regulated by microtubule-associated proteins (MAPs), kinesins, and dyneins facilitating structure and movement.

• Microfilaments: Thin, flexible strands of actin. They are essential for cell motility, muscle contraction, and cytokinesis. Actin polymerizes with ATP and depolymerizes with ADP, showing dynamic behavior.

• Intermediate Filaments: Rope-like structures providing structural support and resilience to cells, anchoring desmosomes and forming the nuclear lamina. Phosphorylation regulates their behaviour.

Cell Signaling

• Cell signaling involves specificity, conformational change, and complex integration to achieve a diverse range of responses.

1. Specificity: Receptor-ligand interactions exhibit high affinity, saturation, and reversibility. 2. Short and Long-Range Signaling: Signals are transmitted locally (paracrine, juxtacrine) or through large distances (endocrine). 2. Calcium (Ca²⁺) release: Released from plasma membranes, ER or sarcoplasmic reticulum through gated channels (voltage-gated channels, IP3 or ryanodine receptors). 3. GPCRs: G-protein coupled receptors initiate cellular responses by activating GTP-dependent signaling cascades. 4. Enzyme-linked receptors: Often tyrosine kinases (e.g., EGF-R), which phosphorylate multiple targets within cells after ligand binding. 5. Cytosolic Receptors: Intracellular receptors bind lipid-soluble signals (e.g., steroids, lipid second messengers, cAMP).

Techniques

• Affinity Chromatography: Isolating proteins based on their specificity for a particular ligand. This method is employed to purify proteins from a sample

• Expression Cloning: Cloning potentially useful genes from a genome library into expression vectors, leading to identification of gene products.

• FRET (Fluorescence Resonance Energy Transfer): This method is used to measure close proximity of proteins, or direct interactions, through energy transfer between fluorescent labels.