Case 6: Protein Destinations PDF

Case 6: Where do the proteins end up? 1. How do proteins fold? Primary structure - The sequence of amino acids in a polypeptide chain - Determined by the DNA of the gene that encodes for the protein - A change in the gene's DNA sequence may lead to a change in the amino acid...

Case 6: Where do the proteins end up? 1. How do proteins fold? Primary structure - The sequence of amino acids in a polypeptide chain - Determined by the DNA of the gene that encodes for the protein - A change in the gene's DNA sequence may lead to a change in the amino acid sequence of the protein. Even changing just one amino acid in a protein’s sequence can affect the protein’s overall structure and function. How many proteins can you make with 20 amino acids? let's make a protein consisting of 60 amino acids: 20^60 options: ~ 10^78 different proteins Secondary structure - Local folded structures that form within a polypeptide due to interactions between atoms of the backbone. (The backbone just refers to the polypeptide chain apart from the R groups – so all we mean here is that the secondary structure does not involve R group atoms.) - Most common: α helix & β pleated sheet - Held in shape by hydrogen bonds In an α helix, the carbonyl (C=O) of one amino acid is hydrogen bonded to the amino H (N-H) of an amino acid that is four down the chain. (E.g., the carbonyl of amino acid 1 would form a hydrogen bond to the N-H of amino acid 5.) This pattern of bonding pulls the polypeptide chain into a helical structure that resembles a curled ribbon, with each turn of the helix containing 3.6 amino acids. The R groups of the amino acids stick outward from the α helix, where they are free to interact In a β pleated sheet, two or more segments of a polypeptide chain line up next to each other, forming a sheet-like structure held together by hydrogen bonds. The hydrogen bonds form between carbonyl and amino groups of backbone, while the R groups extend above and below the plane of the sheet. The strands of a β pleated sheet may be parallel, pointing in the same direction (meaning that their N- and C-termini match up), or antiparallel, pointing in opposite directions (meaning that the N-terminus of one strand is positioned next to the C-terminus of the other). - Many proteins contain both α helices and β pleated sheets, though some contain just one type of secondary structure (or do not form either type) Tertiary structure - The overall three-dimensional structure of a polypeptide Hold together: - Covalent bonds: disulfide bridges are much stronger than the other types of bonds that contribute to tertiary structure. They act like molecular "safety pins," keeping parts of the polypeptide firmly attached to one another. - H-bridges - Salt bridges - Hydrophobic interactions: in which amino acids with nonpolar, hydrophobic R groups cluster together on the inside of the protein, leaving hydrophilic amino acids on the outside to interact with surrounding water molecules. - Metal bridges: between 2 side chains with similar charge Quaternary structure - Some proteins are made up of multiple polypeptide chains, also known as subunits → come together → quaternary structure - Hemoglobin: important one - Another example is DNA polymerase, an enzyme that synthesizes new strands of DNA and is composed of ten subunits - In general, the same types of interactions that contribute to tertiary structure (mostly weak interactions, such as hydrogen bonding and London dispersion forces) also hold the subunits together to give quaternary structure. Protein folding The hydrophobic effect: non-polar amino acids cluster together to avoid water, releasing water molecules into the surrounding environment and increasing the system's entropy. This is the main driver of protein folding, ensuring that the proteins achieve their intricate, functional forms. Forces stabilizing tertiary structure (overall fold) - Hydrophobic interactions: Non-polar amino acid side chains tend to cluster together inside the protein, away from the aqueous environment. This clustering is driven by the hydrophobic effect, which is a major force in stabilizing the protein's structure. The driving force behind this effect is entropy: as hydrophobic residues aggregate, water molecules are released from their structured arrangements around these residues, increasing the entropy of the system. - Hydrogen bonds: These form between polar side chains and contribute significantly to the protein's stability. Hydrogen bonds can occur between side chains and the backbone (as occurs with secondary motifs), or between side chains themselves. - Ionic bonds (salt bridges): These form between positively charged (basic) and negatively charged (acidic) side chains. Ionic bonds are strong and contribute to the overall stability of the protein structure. - Disulfide bonds: These covalent bonds form between the sulfur atoms of two cysteine residues. Disulfide bonds provide significant stabilization, particularly in extracellular proteins. - Van der Waals forces: These weak interactions occur between all atoms that are in close proximity. They help to fine-tune protein structure and stability. Entropic contributions to folding Protein folding is driven by a balance of enthalpic and entropic changes: - Conformational entropy: As a protein folds, it adopts a more ordered structure, which decreases its conformational entropy. However, this loss is offset by other entropic gains. - Solvent entropy: The hydrophobic effect plays a critical role here. When hydrophobic side chains cluster together in the interior of the protein, water molecules that were structured around these hydrophobic residues are released into the bulk solvent. This release increases the entropy of the surrounding water molecules, which is a driving force for the folding process. Forces stabilizing quaternary interactions - Hydrophobic Interactions: Subunits often interface through hydrophobic regions to minimize exposure to the aqueous environment, driven by entropy. - Hydrogen Bonds: These stabilize the interfaces between subunits. - Ionic Bonds: These can form between charged residues at the interfaces of subunits. - Disulfide Bonds: In some multi-subunit proteins, disulfide bonds can link different polypeptide chains. Guided Protein Folding The process of folding is guided by the protein's primary sequence but is often assisted by molecular chaperones. Chaperones, including heat shock proteins (HSPs) and chaperonins, play crucial roles in ensuring correct protein folding. Chaperone proteins are needed to assist protein folding because the cellular environment can be crowded and stressful, which can cause nascent or misfolded proteins to aggregate or fold incorrectly. Chaperones help by stabilizing these proteins, preventing aggregation, and providing an optimal environment for proper folding, ensuring that proteins achieve their correct functional conformations. - Molecular Chaperones: These proteins bind to nascent or partially folded polypeptides, preventing improper interactions that can lead to aggregation or misfolding. For example, HSP70 chaperones bind to hydrophobic regions of nascent proteins, preventing aggregation and facilitating correct folding. Protect from degradation, they’re there to help folding. - Chaperonins: These are large, cylindrical complexes that provide a protected environment for protein folding. A well-known example is the GroEL/GroES system in bacteria. GroEL is a barrel-shaped protein that encapsulates the polypeptide, while GroES acts as a cap. This isolation prevents aggregation and allows the protein to fold properly within the chamber. After folding, the protein is released, and the chaperonin complex is ready to assist with another substrate. If protein folding is not correct → misfolded proteins start to accumulate → protein aggregation → degenerative diseases Protein folding in mitochondria Proteins that are meant to go into the mitochondrial matrix have to pass through both the inner and outer membranes of the mitochondria in an unfolded form. This movement is closely linked to the process of unfolding proteins in the cytosol and refolding them once they reach the matrix. To help bring proteins from the cytoplasm into the mitochondria, cells use various molecular chaperones and folding helpers. One key player inside the mitochondria is mitochondrial Hsp70 (mt-Hsp70), which has several roles. First, mt-Hsp70, along with other proteins, binds to the incoming protein chains to ensure they move in one direction during translocation and helps refold them. Some proteins also need extra help from chaperonins of the Hsp60/Hsp10 family, which fold the proteins inside a special chamber. Proper interaction with these chaperones is important not just for folding and assembly, but also for processing, sorting, and breaking down proteins inside the mitochondria. https://pubmed.ncbi.nlm.nih.gov/9067800/#:~:text=Protein%20folding%20occurs%20within% 20the,sorting%2C%20and%20degradation%20of%20proteins. - Chaperones can also help with unfolding 2. What are the post translational modifications? Here’s a list of common post-translational modifications (PTMs) of proteins based on scientific knowledge: 1. Phosphorylation Addition of a phosphate group (usually to serine, threonine, or tyrosine residues) by kinases. This modification regulates many cellular processes, including signal transduction and protein activity. Add phosphate on protein → protein kinase do that → activates phosphatase dephosphorylates → deactivates (Can also be other way around) 2. Glycosylation Attachment of carbohydrate groups to asparagine (N-linked glycosylation) or serine/threonine residues (O-linked glycosylation). Glycosylation is critical for protein folding, stability, and cell-cell recognition. - Protein glycosylation underpins the ABO blood group system - Defects in protein glycosylation can cause congenital disorders - Used by viruses to hide the viral protein for immune recognition by the host 3. Proteolysis Cleavage of specific peptide bonds within a protein. This modification is important for activating proteins, like the conversion of proenzymes to their active forms (e.g., proinsulin to insulin). (delta notch signaling) 4. Hydroxylation Addition of a hydroxyl group, typically to proline or lysine residues. This modification is essential in collagen biosynthesis and the response to hypoxia (e.g., hypoxia-inducible factor (HIF) regulation). Fold tighter (collagen skin) → hydroxyl groups in proline form bridges for tighter collagen fibers. 5. Disulfide Bond Formation Covalent linkage between two cysteine residues, forming a disulfide bond. This modification is crucial for the stability and structural integrity of many proteins. Strong bonds. 6. Ubiquitination (degradation) Addition of ubiquitin, a small regulatory protein, to lysine residues. Ubiquitination often targets proteins for degradation via the proteasome but also regulates other cellular processes like endocytosis and DNA repair. Methylation Acetylation Lipidation 3. How are proteins transported to their final destination? Proteins can follow various routes depending on their cellular function and targeting signals. The key stages include synthesis in the cytosol, potential entry into the endomembrane system (ER, Golgi), and final targeting to various cellular compartments or secretion. Based on their amino acid sequence proteins are directed to their destination: Protein Pathways and Destinations: 1. Protein Synthesis in the Cytosol All proteins are synthesized by ribosomes, either free in the cytosol or associated with the rough endoplasmic reticulum (ER). The destination depends on whether the protein has a signal sequence or localization signal. 2. Cytosolic Proteins Proteins with no targeting sequence remain in the cytosol. These can perform various functions within the cytoplasm, such as metabolic enzymes, cytoskeletal proteins, etc. 3. Post-Translational transport Pathways - Nucleus: Proteins with a nuclear localization signal (NLS) are imported into the nucleus through nuclear pore complexes. Examples: transcription factors, histones. - Mitochondria: Proteins with a mitochondrial targeting sequence are imported into mitochondria via translocases (TOM/TIM complexes) across outer and inner membranes. These proteins are directed to the outer membrane, inner membrane, intermembrane space, or matrix. - Peroxisomes: Proteins with a peroxisomal targeting signal (PTS1 or PTS2) are imported into peroxisomes, where they function in fatty acid metabolism and detoxification. Proteins need to be folded to get into the peroxisomes. 4. Co-Translational transport: Endoplasmic Reticulum (ER) Secretory and Membrane Proteins: Proteins with an N-terminal signal peptide are directed to the ER during translation. The signal recognition particle (SRP) binds the signal peptide and directs the ribosome to the ER membrane. The proteins enter the ER co-translationally and either remain membrane-bound or enter the ER lumen. ER-Resident Proteins: Proteins with an ER retention signal (e.g., KDEL) remain in the ER and function in protein folding or modification (e.g., chaperones, folding enzymes). Golgi Apparatus: Proteins destined for secretion, plasma membrane, or lysosomes are transported from the ER to the Golgi for further processing and sorting. Secretory Pathway:  Proteins are processed in the Golgi and packed into secretory vesicles, which move toward the plasma membrane. Constitutive Secretion: Proteins are continuously secreted without further regulation (e.g., collagen). Regulated Secretion: Proteins are stored in secretory granules and released in response to specific signals (e.g., hormones, neurotransmitters). Plasma Membrane: Transmembrane proteins are inserted into the plasma membrane, where they perform roles in cell signaling, transport, and adhesion. Lysosomes: Proteins tagged with mannose-6-phosphate in the Golgi are targeted to lysosomes, where they act as enzymes for degradation of macromolecules. 5. Endocytic and Autophagic Pathways Endocytosis: Proteins on the plasma membrane can be internalized and targeted to endosomes, then delivered to lysosomes for degradation or recycled back to the plasma membrane. Autophagy: Cytosolic proteins and organelles can be sequestered into autophagosomes and delivered to lysosomes for degradation, contributing to cellular homeostasis. Summary of Major Protein Destinations: - Cytosol: Free proteins without signal sequences. - Nucleus: Proteins with nuclear localization signals. - Mitochondria: Proteins with mitochondrial targeting sequences. - Peroxisomes: Proteins with peroxisomal targeting sequences. - Endoplasmic Reticulum (ER): ~ ER-resident proteins. - Golgi apparatus for further modification and sorting. ~ Secretory proteins (constitutive or regulated secretion). ~ Plasma membrane proteins. ~ Lysosomal enzymes. Post-translational transport Nucleus (gated) Gated/pore transport → is transport between ‘equal compartments’ Transport via a pore → nuclear pore Mitochondria (translocaters) Transport via translocators → transport between compartments with different environments The Msf1 (Mitochondrial import stimulation factor 1), also known as mitochondrial sorting factor 1, is a chaperone protein involved in the import of proteins into the mitochondria, specifically targeting precursor proteins to the outer mitochondrial membrane. It plays a critical role in recognizing and guiding proteins with specific mitochondrial targeting signals to the appropriate translocase complexes. In summary, Msf1 is a mitochondrial chaperone that recognizes and delivers certain mitochondrial outer membrane proteins to the TOM complex, ensuring their correct import and preventing misfolding or aggregation during the process. Co-Translational Transport Endoplasmic Reticulum Some ribosomes are free floating in the cytoplasm, and others are attached to the membrane of the rough endoplasmic reticulum (rER). These ribosomes have the same structure but make different proteins. - ER-bound ribosomes synthesize all secreted proteins, plasma membrane proteins, and the proteins of ER, Golgi apparatus, and lysosomes. - The proteins of cytoplasm, nucleus, and mitochondria are synthesized both by free-floating ribosomes and by ribosomes that are attached to the ER. Proteins that have to be processed through the ER start with a signal sequence of about 20 to 25 mainly hydrophobic amino acid residues at the amino end. This is the part of the protein that is synthesized first by the ribosome. As soon as it emerges from the ribosome, the signal sequence binds to a cytoplasmic signal recognition particle (SRP), which contains a small RNA molecule of about 300 nucleotides (the 7SL RNA) and six protein subunits. Binding of the SRP halts translation. Translation resumes only when the SRP-signal sequence-ribosome complex binds to an SRP receptor on the ER membrane The SRP receptor brings the ribosome in contact with a protein translocator, a donut-shaped protein in the ER membrane. The tunnel on the large ribosomal subunit from which the growing polypeptide emerges is placed on the central hollow of the protein translocator while the SRP detaches. A pore opens in the translocator, through which the polypeptide passes into the lumen of the rough ER. The signal sequence is no longer required beyond this stage. It is cleaved off by a signal peptidase on the inner surface of the ER membrane. You can alternate the stop/start sequences to have membrane proteins that go through the membrane Free ribosome and ER-bound ribosomes: Golgi apparatus (Vesicular transport) There are different types of vesicles, different types of coat proteins: clathrin, COPI, COPII. - COPII coating takes place in ER → allow fusion with cis part of golgi - COPI sends back to golgi because of faulty modification, or sometimes proteins need to be modified in golgi and then sent back to ER to be functional inside the ER. Also for exocytosis. - Clathrin for endocytosis Budding process with endocytosis: Phosphatidyl-inositol-phosphates (PIP), you can modify the inositol group with phosphates → can be used by other proteins to recognize → right transportation Rab proteins: proteins connected to vesicle for specific location Job of a protein 1. Structure (hair, nails) 2. Enzymes 3. Movement (muscle) 4. Transport (O2, membrane transport) 5. Hormones (e.g. insuline) 6. Protection (blood clothing, antibodies) 7. Nutrients 8. Regulation 4. Why and how proteins are degraded (degradation)? Protein degradation is a vital process that ensures cellular homeostasis by removing damaged, misfolded, or unneeded proteins. This process can occur via several pathways, often involving chaperones, proteasomes, and the unfolded protein response (UPR). 1. Chaperones: Protein Quality Control - Chaperones are proteins that assist in the proper folding of other proteins. They monitor the cell for misfolded or unfolded proteins, preventing aggregation and aiding in refolding when possible. Common chaperones include Hsp70 and Hsp90. - If a protein is irreparably damaged or cannot fold correctly despite chaperone assistance, chaperones tag these misfolded proteins for degradation. - Example: Chaperones can recognize misfolded proteins and shuttle them to degradation pathways by marking them with ubiquitin, a signal for protein destruction. 2. Proteasomes: The Protein Destruction Machinery - The proteasome is a large protease complex responsible for degrading proteins tagged with ubiquitin (a process called ubiquitination). - Proteins destined for degradation are marked by the covalent attachment of ubiquitin chains, a signal recognized by the 26S proteasome. These proteins are unfolded and threaded into the proteasome, where they are degraded into smaller peptides. Ubiquitin-Proteasome Pathway (UPP): This pathway is central to protein degradation. Ubiquitin ligases attach ubiquitin molecules to proteins that are damaged, misfolded, or no longer needed, targeting them for degradation by the proteasome. Works in the cytosol. Chaperone involvement: Chaperones often facilitate the recognition of damaged proteins, assisting in their delivery to ubiquitin ligases or directly to the proteasome for degradation. 3. Unfolded Protein Response (UPR): Cellular Stress Management  - The unfolded protein response (UPR) is activated when there is an accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), leading to ER stress. This can happen due to various cellular stresses, like nutrient deprivation or oxidative stress. - The UPR aims to restore protein folding capacity by: Upregulating chaperone production: More chaperones are synthesized to help refold misfolded proteins. Degrading misfolded proteins: If refolding is unsuccessful, the UPR activates degradation pathways, such as ER-associated degradation (ERAD), where misfolded proteins are retro-translocated to the cytosol and degraded by the ubiquitin-proteasome system. - If UPR cannot restore normal protein folding, it can trigger cell death (apoptosis) to prevent further damage. Overview of Protein Degradation Pathways: 1. Chaperones identify misfolded or damaged proteins and attempt refolding. If unsuccessful, they mark proteins for degradation. 2. Proteasomes degrade ubiquitin-tagged proteins into smaller peptides, a major pathway for removing damaged or short-lived proteins. 3. In cases of ER stress, the UPR is activated to restore normal protein folding, or, if necessary, degrade excess misfolded proteins through the proteasome. If chaperones can't release themselves from the protein → can cause issues as well. 5. What are the diseases? Apply info to case. Gaucher’s disease Gaucher’s disease is a genetic disorder caused by a deficiency in the enzyme glucocerebrosidase (also called acid β-glucosidase), which is responsible for breaking down a fatty substance called glucocerebroside. When this enzyme is deficient or not functioning properly, glucocerebroside accumulates in certain cells, particularly macrophages, which are part of the body's immune system. These enlarged, lipid-filled macrophages are called Gaucher cells. I-Cell disease I-Cell disease is a rare, inherited lysosomal storage disorder caused by a defect in the process that tags enzymes for transport to lysosomes. This results in the improper degradation of cellular waste, leading to the accumulation of undigested substances (such as lipids and carbohydrates) within lysosomes, which then form dense inclusions in cells, hence the name “inclusion cell” (I-Cell) disease. - The genetic defect occurs in the GNPTAB gene, which encodes for the enzyme N-acetylglucosamine-1-phosphotransferase. This enzyme is crucial for tagging certain lysosomal enzymes with a mannose-6-phosphate (M6P) marker, which directs them to lysosomes. - In I-Cell disease, the mannose-6-phosphate tag is missing from lysosomal enzymes due to the defective GNPTAB gene. - Without the M6P tag, these enzymes are not properly directed to the lysosomes. Instead, they are secreted outside the cell into the extracellular space, leaving the lysosomes unable to degrade waste material. - As a result, undigested materials like glycoproteins and glycolipids accumulate within lysosomes, leading to the formation of "inclusion bodies" in cells. - This buildup disrupts normal cellular function, causing widespread tissue and organ damage.