BIOL340: Cell & Molecular Biology Intracellular Proteostasis Lecture 5 PDF

BIOL340: Cell & Molecular Biology Intracellular Proteostasis Week 3 Lecture 5 11th March, 2024 Anuk Indraratna [email protected] 1 Learning outcomes 1. Explain the processes and importance underlying protein conformation. 2. Describe, with an example, the activity of chaperone proteins. 3. Explain the...

BIOL340: Cell & Molecular Biology Intracellular Proteostasis Week 3 Lecture 5 11th March, 2024 Anuk Indraratna [email protected] 1 Learning outcomes 1. Explain the processes and importance underlying protein conformation. 2. Describe, with an example, the activity of chaperone proteins. 3. Explain the ubiquitin-proteasome system generally, and with regard to the endoplasmic reticulum-associated degradation pathway. 4. Briefly outline the unfolded protein response and heat shock response. 5. Briefly describe the role of the aggresome in protein homeostasis. 6. Give an overview of all major mechanisms involved in intracellular proteostasis. 7. Describe, with examples, the importance of protein misfolding in disease. 2 Lecture outline 1. Overview of protein structure 2. Protein folding and misfolding 3. Mechanisms of internal proteostasis A. Chaperones B. Protein degradation C. ERAD pathway D. Cellular responses E. Aggresomes 4. Diseases of protein misfolding 3 Lecture outline 1. Overview of protein structure 2. Protein folding and misfolding 3. Mechanisms of internal proteostasis A. Chaperones B. Protein degradation C. ERAD pathway D. Cellular responses E. Aggresomes 4. Diseases of protein misfolding 4 Proteins: nature’s incredible molecule  Proteins are most of the cell’s dry mass  Perform a lot of the cell’s functions  400 g protein turnover a day; 1.5 x 1028 molecules 5 Levels of protein structure 6 Haemoglobin 7 Protein domains  Distinct structural or functional units within the protein — Typically responsible for a specific interaction — Referred to with regard to their ligand-binding behaviour, or shape — E.g. alpha-helical, N-terminal, glucose-binding domain  Certain domains are described by conserved function — Kinase domain, zinc fingers, SH2 domains  Domains are modular — Constituent, somewhat independent units that comprise larger proteins — ~40-350 amino acids in length that folds independently 8 Lecture outline 1. Overview of protein structure 2. Protein folding and misfolding 3. Mechanisms of internal proteostasis A. Chaperones B. Protein degradation C. ERAD pathway D. Cellular responses E. Aggresomes 4. Diseases of protein misfolding 9 Protein folding Folding Folding Unfolded Folded = functional Folding Folding Folded = functional 10 Protein folding 11 Self-assembly and disassembly  Under denaturing conditions, structure is compromised — pH, temperature, chemical, ionic strength, radiation etc. — Destructive processes need to be contained (lysozymes, peroxisomes)  Loss of secondary and tertiary structure, but not primary — Hydrogen & ionic bonds, disulphide bridges, hydrophobic interactions etc. are disrupted — Peptide bonds are unaffected  Denaturation can be reversible — If no covalent modifications occur, denaturation may be reversible — When normal conditions are restored, the protein may regain its structure and function 12 Protein folding thermodynamics  Levinthal’s paradox: protein folding must be non-random — Vast number of conformations are theoretically probably E.g. 100 amino acid peptide = 3^100 conformations — Random ‘trial-and-error’ would take an eternity — However, proteins fold rapidly within milliseconds to seconds  Protein folding is guided through energetically favourable transition states — Unfolded state is high-energy — Progresses towards a state of decreased free energy Free rotation around single bonds in peptide backbone 13 Protein folding thermodynamics 14 Protein folding pathways 1. Hydrophobic collapse — Hydrophobic moieties want to avoid water and associate with other hydrophobic moieties — Brings hydrophobic areas together, decreasing the system’s overall energy 2. Nucleation of folded domains — Formation of small, stable regions of secondary structure — Promotes hydrophobic collapse  These processes happen together and driven by each other 15 Green - Hydrophilic Red - Hydrophobic 16 Protein misfolding and aggregation Native Ordered state Disordered state Ordered state Disordered state Unfolded Fibril nucleus Amorphous aggregate Amorphous aggregation pathway: fast, disordered aggregates Amorphous precipitate Amyloid fibril pathway: slow, highly ordered aggregates Toxic oligomer Fibril 17 Lecture outline 1. Overview of protein structure 2. Protein folding and misfolding 3. Mechanisms of internal proteostasis A. Chaperones B. Protein degradation C. ERAD pathway D. Cellular responses E. Aggresomes 4. Diseases of protein misfolding 18 Proteostasis = protein homeostasis 19 Chaperones  Class of proteins that facilitate folding of client proteins — Specifically recognize and bind to non-native proteins via exposure of hydrophobic surfaces — Prevent inappropriate aggregation; covers regions of hydrophobicity — Varied mechanisms of action — Varied specificity  Holdases — Work to stabilize protein and prevent aggregation — ATP-independent; don’t actively refold client  Foldases — Facilitate correct folding of clients — ATP-dependent https://physicallensonthecell.org/sites/default/fil es/chapcycle.gif 20 21  Heat shock protein 70 (Hsp70) is a typically acts upon newly synthesised polypeptides  Has both holdase and foldase activities  Shields hydrophobic regions from inappropriate interactions  ATP-dependent binding of polypeptide  ATP-dependent release for proper folding  Mechanism is not fully understood still 22 Chaperone-mediated autophagy  System that involves recognition of specific motif: KFERQ — About 30% of cytosolic proteins have this motif — Under stress conditions, becomes accessible to heat shock cognate 70 (Hsc70)  Hsc70 binds to the protein via this motif and directs it towards the lysosome — The complex interacts with LAMP-2A receptor — Lysosome-Associated Membrane Protein  LAMP2A forms a large multimeric channel  Hsc70 unfolds the protein as it translocates — Protein is threaded through the complex into lysosome — Proteolytic degradation occurs within lysosome 23 Lecture outline 1. Overview of protein structure 2. Protein folding and misfolding 3. Mechanisms of internal proteostasis A. Chaperones B. Protein degradation C. ERAD pathway D. Cellular responses E. Aggresomes 4. Diseases of protein misfolding 24 Protein degradation  If proteins cannot be refolded, it must be disposed of  Two main compartments responsible for degradation — Lysosome — Proteasome  Misfolded proteins tagged and delivered to proteasome  Relies on ubiquitin 25 Ubiquitin-proteasome system (UPS) 1. Ubiquitin ligase recognizes non-native protein and attaches chains of ubiquitin (small protein). 2. Poly-ubiquitinated protein binds to cap of proteasome. 3. Ubiquitin is removed; protein begins to unfold and enters chamber. 4. Within the ‘hollow’ chamber, the protein is degraded into small peptide fragments. 5. Peptide remnants are released into cytosol. 26 27 Lecture outline 1. Overview of protein structure 2. Protein folding and misfolding 3. Mechanisms of internal proteostasis A. Chaperones B. Protein degradation C. ERAD pathway D. Cellular responses E. Aggresomes 4. Diseases of protein misfolding 28 Endoplasmic reticulum associated protein degradation (ERAD)  A sub-category of the UPS where misfolded or unassembled proteins are recognised within the ER  Early recognition prevents accumulation or inappropriate trafficking to other locations  If enough material accumulates in ER, the unfolded protein response (UPR is activated) 29 Cellular stress responses  Unfolded protein response (UPR) aims to restore homeostasis within the ER — — — —  Reducing the load of proteins entering the ER Upregulating chaperone expression Upregulated ERAD UPR can ultimately trigger apoptosis (programmed cell death) Heat shock response — Triggered by increase in temperature or other stress (hypoxia, oxidative stress, heavy metals etc.) — Heat Shock Factor 1 (HSF1) forms trimer and enters nucleus — Binds to heat shock elements (promoters of heat shock genes) — Rapid upregulation of Hsp 30 Lecture outline 1. Overview of protein structure 2. Protein folding and misfolding 3. Mechanisms of internal proteostasis A. Chaperones B. Protein degradation C. ERAD pathway D. Cellular responses E. Aggresomes 4. Diseases of protein misfolding 31 Aggresomes  Forms other degradation mechanisms are overwhelmed, inhibited, or otherwise ineffective  Accumulation of misfolded / aggregated protein — Core of aggregated protein — Surrounded by cage of intermediate filaments — Sequesters the aggregate and recruits other elements of proteostasis machinery  Essentially serves as temporary storage site where problematic aggregates can be deposited 32 Lecture outline 1. Overview of protein structure 2. Protein folding and misfolding 3. Mechanisms of internal proteostasis A. Chaperones B. Protein degradation C. ERAD pathway D. Cellular responses E. Aggresomes 4. Diseases of protein misfolding 33 Diseases of protein misfolding  Consistent accumulation of misfolded protein can lead to disease  Insoluble fibrils or plaques interrupt cellular function — Build up in central nervous system leads to neurodegenerative disease such as Alzheimer’s 34 Native Ordered state Disordered state Ordered state Disordered state Unfolded Fibril nucleus Amorphous aggregate Amorphous aggregation pathway: fast, disordered aggregates Amorphous precipitate Amyloid fibril pathway: slow, highly ordered aggregates Toxic oligomer Fibril 35 Diseases of protein misfolding Protein involved Disease(s) Amyloid–β Alzheimer’s disease tau Alzheimer’s disease α-Synuclein Parkinson’s disease, Lewy body dementia Amylin Diabetes type 2 SOD1, TDP-43 Amyotrophic Lateral Sclerosis (MND) Haemodialysis-related amyloidosis β2-Microglobulin Amyloid-A Reactive amyloidosis Haemoglobin Sickle cell anaemia Huntington Huntington’s disease PrP Creutzfeldt-Jakob disease Androgen receptor Spinobulbar muscular atrophy Ataxins 1,2 & 3 Spinocerebellar ataxia’s 1,2 & 3 Ten other proteins Systemic and cerebral hereditary amyloidosis 36 Protein misfolding in Alzheimer’s  Protein misfolding disorder; amyloidosis — Specifically amyloid-beta (Aβ) plaques and tau protein tangles  Amyloid-beta aggregation in neuronal cells leads to cell death — Initially, Aβ peptide fragments form soluble oligomers which are toxic to neurons — Further aggregation leads to insoluble, extracellular plaques  Tau protein is also involved — Functional tau stabilizes microtubules in neurons — Abnormally high degrees of phosphorylation (hyperphosphorylation) — This leads to reduced affinity for microtubules and misfolding — Misfolded tau aggregated to form helical filaments and neurofibrillary tangles — Neuronal transport system is disrupted, leading to cell dysfunction and death  Accumulation of ab plaques and tau tangles leads to neural death — Extracellular plaques lead to inflammation which accelerates Alzheimer’s process — Tissue destruction in various parts of brain lead to various cognitive deficits 37 Parkinson’s Alzheimer’s 38 ALS (MND) is associated with inclusions FUS immunoreactivity Human ALS Human ALS Deng et al., ANN NEUROL 2010;67:739–748 Human fALS TDP-43 immunoreactivity Mackenzie et al., Ann Neurol 2007;61:427–434 ALS mice SOD1 immunoreactivity Chattopadhyay and Valentine, Antiox & Redox Signal 11(7), 2009 39 Learning outcomes 1. Explain the processes and importance underlying protein conformation. 2. Describe, with an example, the activity of chaperone proteins. 3. Explain the ubiquitin-proteasome system generally, and with regard to the endoplasmic reticulum-associated degradation pathway. 4. Briefly outline the unfolded protein response and heat shock response. 5. Briefly describe the role of the aggresome in protein homeostasis. 6. Give an overview of all major mechanisms involved in protein homeostasis. 7. Describe, with examples, the importance of protein misfolding in disease. 40 Example quiz questions 1. Describe in detail the mechanisms of internal proteostasis. [10 marks] 2. Explain, with an example, how protein misfolding can lead to disease. [5 marks] 3. Explain how nascent polypeptides eventually fold into mature proteins. [5 marks] 4. Compare the heat shock response and the unfolded protein response with regard to its triggers and outcomes. [6 marks] 41