1. MedSoc Teaching

2. Physiology Vs Pathology
Protein turnover
and
catabolic disease.
Protein turnover is the physiologic function. When this normal homeostatic mechanism goes wrong then we have abnormal cell behaviour therefore disease. In a structured essay you should first say what is normal, which will later help you explain why something is abnormal.
Physiology Vs Pathology

3. Sample exam question
Describe the major pathways of intracellular protein degradation (40%) and discuss their contribution to neuronal death and survival in neurodegenerative disease (60%).

4. Brief summary/Conclusion
Introduction
State the importance of proteostatic control in cellular homeostasis.
Main text
UPS 1) definition 2) importance 3) diagram
2) Autophagy 1) definition 3) importance 4) diagram 5) Atg proteins
3) Proteostasis: Balance between protein synthesis- protein degradation
4) Flow diagram of progression to protein aggregation/pathology
5) Define proteinopathy and relate to neurodegenerative disease giving specific examples
6) Mention genes mutated in NDD ie parkin, UCH-L1, VCP
7) Expand on VCP (involved in both UPS and autophagy)
Brief summary/Conclusion +
Clinical relevance
Therapeutic implications
Q.15

5. Protein turnover
State the importance of proteostatic control in cellular homeostasis.
2) Describe in words and using diagrams the processes of UPS and autophagy clearly demonstrating the role of ubiquitin in each process .
3) Know how to compare the two processes

6. -Proteins dictate a cell’s behaviour.
-Protein turnover is a dynamic process which needs to be tightly regulated.
-Deregulated protein behaviour =
deregulated cell behaviour.
-Abnormal cell behaviour= DISEASE .
WHAT YOU NEED TO KNOW IS THAT PROTEINS WILL DICTATE THE FATE OF THE CELL. IF THERE IS NORMAL PROTEIN BEHAVIOUR IN THE CELL THEN THERE IS NORMAL CELL BEHAVIOUR. IF THERE IS DERGEULATED PROTEIN BEHAVIOUR IN THE CELL THEN THERE IS DEREGULATED CELL BEHAVIOUR. A CELL WHICH BEHAVES ABNORMALLY = DISEASE
Protein behaviour directs cellular behaviourDeregulated protein behaviour in turn means deregulated cell behaviour. When a cell behaves abnormally this indicated disease. Cellular protein homeostasis= proteostasis requires a balance between protein synthesis and degradation therefore protein turnover need to be TIGHTLY REGULATED- Who regulates this behaviour? UPS AND AUTOPHAGY. REMOVAL OF PROTEIN ABUNDANCE ALLOS FOR PROPER CELL DIVISION, DNA REPAIR, DIFFERENTIATION AND GROWTH. CONSEQUENTLY A FAULTY PROTEOLYTIC SYSTEM CAN ALTER CELL FATE AND BRING ABOUT PATHOGENESIS
Proteins turnover is a dynamic process, proteins constantly get degraded and synthesised according to the cell’s needs
Proteolytic pathways act as the cell's quality control systems by disposing of damaged, misshapen, and excess proteins

7. STATE the Function & Importance of UPS
Multisubunit proteolytic pathway (operates in nucleus and cytoplasm) which uses Ubiquitin, a small molecule of 76 amino acids, to tag protein substrates (soluble short lived intracellular proteins) for degradation by the 26S proteosome system.
Due to the importance of its function, (has the authority to KILL proteins) it has evolved to be highly selective and specific to its degradation substrates so not to abuse its authority.
2 E1 enzymes -non specific and activate ubiquitin for all modifications, can activate E2 enzymes which then selectively attach to the specific E3 enzyme (x1000). The unique E2-E3 combinations will selectively target the protein to be degraded.
*note ubiquitin is a 76 amino acid molecule found ubiquitously in the cell. Mention that there are 2 E1 enzymes which are non specific and activate ubiquitin for all modifications, which can activate E2 enzymes which can then selectively attach to the specific E3 enzyme of which there are approximately The unique E2-E3 combinations will then selectively target the protein to be degraded.

8. DRAW an annotated diagram illustrating the UPS
Ub b
26S
proteasome Ub
DUB E2
DRAW an annotated diagram illustrating the UPS 1) 4) 5) 6) 3) E3
ATP
19 S
20S
ATP
Lys
Figure 3
Schematic diagram illustrating the multi-step enzymatic pathway of the UPS
To start with, Ub is covalently activated by E1-Ub activating enzyme with the formation of an thioster bond between Cterminal glycine (Ub) and internal cysteine E1 (in an E dependent process)
Activated Ub is then transferred to an E2-Ub conjugating enzyme
In the meantime, E3-Ub ligase enzyme recognises and interacts with substrate
E3-Ub ligase mediates transfer of Ub tag from E2 enzyme to substrate. The first Ub moiety is transferred to a lysine residue of the protein substrateto give a covalently bonded polyubiquitinated chain in which Ub are attatched amongsth them at lysine48 residue
Polyubiquitinated chain acts as cellular signal for irreversible destruction of tagged protein by 26S proteosome system to give functionless oligomers
Deubiquitinating (DUB) enzyme breaks down polyubiquitinated chain to monoubiquitins which are recycled for continuation of the UPS cycle
Diagram drawn by Eilana Kalakouti

9. 19S Recognises Unfolds Removes polyubiquitin chain Internalize/α 1 2 3 4 5 6 7
Internalize/
degrade substrate to inactive amino acids
Catalytic core:
trypsin,
chymotrypsin, postglutamyl peptidyl β 1 2 3 4 5 6 7 1 2 3 4 5 6 7 β α 1 2 3 4 5 6 7
26S proteosome is composed of the 20S core catalytic complex flanked on both sides by the 19S regulatory complexes.
The 20S complex is a stack of 4 rings: 2-α and 2-β organised as a(7)b(7)b(7)a(7). Each chain consists of 7 distinct subunits, the a chains stabilize the b chains and help bind to the 19Scap. b chains carry out the proteolytic activity having 3 catalytic sites: trypsin, chymotrypsin and peptidyl glutamyl. The 20S contains the catalytic domain whereas the19S is responsible for subtrate specificity, recognising the specific substrate, unfoliding it and removing ubiquitin and then passing on to 20S for deactivation of protein into amino acids
- 4 stacked heptameric ring structures . The 2 outer 7a subunits serve as docking domains whereas the inner 2 rings 7β consist the protease active sites and perform the proteolysis reaction

10. STATE the Function & Importance of autophagy
Macroautophagy (mention microautophagy, & chaperone mediated autophagy) : it is a tightly regulated process involved in the degradadation of cells own components ( damaged organelles and long lived proteins) through lysosomal machinery.  
Selective: nutrient rich conditions
Non selective : starvation
Autophagy is a conserved process of self-digestion, ‘self-eating’, which is utilized by cells in all multi-cellular organisms to survive a variety of stressful conditions, including nutrient deprivation, environmental changes, and infection. In addition, autophagy has a key role during development, in prevention of disease, and aging, although paradoxically autophagy may not always be beneficial. Given the complex and wide-ranging role of autophagy, it is perhaps not surprising that it is a highly regulated process.
Non selective: engulfment of pockets of cytoplasm containing proteins and organelles- non specific, starvation induced recycling of bulk, in desperate need of nutrients. Non selective: self-digestion of random, bulky pockets of cytoplasm containing organelles and proteins. Induced in starvation when in desperate need of Energy and nutrients. Inhibited by nutrient-rich conditions.
In a healthy cell it is involved in the maintenance of the cell’s proteoastasis. In a starving cell, it is a major mechanism by which the cell reallocates nutrients from unnecessary processes to more-essential processes.
Selective: degradation of specific tagged organells and proteins inaccesible to the proteosome. Ubiquitin tags protein substrates for selective autophagy recognised by polyubiquitinated chains in which the ubiquiti molecules are conjugated to each other via Lys63 residue (not lys 48 like UPS)
targeted organelles and protein aggregates. Ubiquitin used as a signal of selective autophagy. Polyubiquitin chains joined at Lys63 .
Non specific engulfement of cytoplasm bulk when in desperate need of nutrients

11. Degradation 3 4 2 1 Sequestration
Formation and elongation of double layered isolation membrane 3 4 2 1
Phagophore or isolation membrane:
Double membrane that encloses and isolates the autophagic substrate ie misfolded protein or organelle
Autophagosome:
Double membrane organelle formed upon complete elongation of phagophore
DRAW an annotated diagram illustrating the autophagy process
2 steps in autophagy: The most well-known mechanism of autophagy involves the formation of a membrane around a targeted region of the cell, separating the contents from the rest of the cytoplasm. The resultant vesicle then fuses with a lysozome and subsequently degrades the contents.
Once formed the autophagosomes are transported along microtubules in cell in a dynein dependent way. The outer membrane of the autophagosomes will fuse with an endosome or directly with a lysosome where the autophagic material will be degraded by acidic lysosomal hydrolyses into inactive amino acids.
Induction- formation and elongation of a double layered isolation membrane
Phagophore
Autophagosome
autophagolysosome
Sequestration

12. Mention the autophagy related proteins
>34 proteins required for autophagy –Atg
Their function is almost entirely dedicated to make sure that the autophagosomes have the correct composition and content
Autophagosome induction: Atg1 complex (Atg1, 13 and 17)
Vesicle nucleation: PI3K complex III (consists of cps34, Beclin 1 and UVRAG)
Vesicle elongation and sealing: 2 conjugation systems:
-1st conjugation system involves formation of Atg5-12 conjugate mediated by Atg7 (E1)-Atg10 (E2)
-2nd conjugation system involves Atg8(LC3)-phosphatidylethanolamine (PE) conjugate-Atg7 (E1) and Atg3 (E2)
Autophagy related proteins
Formation of the autophagosome is regulated by a large family of autophagy related proteins grouped according to their function. Atg proteins essential for autophagy!!!Currently, there are over 34 proteins that are required for autophagy. They were first systematically identified in yeast and called Atg (autophagy related) proteins The function of these 34 Atg proteins is almost entirely dedicated to making autophagosomes with the correct composition and content. They are grouped according to theri function
Formation of autophagosome is regulated by a large family of Atg or autophagy related keys. The Atg1 group consists of Atg1, 13,17 proteins and function to control autophagosome induction whereas the PI3K III complex acts to control vesicle nucleation. In addition there are two Ub conjugation like systems which are interconected and mediate vesicle elongation and sealing. The 1st conjugatin system is the Atg5-12 and the 2nd one the PE-LC3 complex. Following formation of the autophagosome atg5-12 complex is removed from the vesicle whereas Atg8/Pe remains in association with it and acts as a scaffold complex helping autophagosome maturation
One key Atg-containing complex is the autophagy-specific class III phosphatidylinositol 3-kinase (PI3P-kinase), which produces PI3P at the site of autophagosome formation . In metazoans, this lipid kinase complex is composed of a kinase, hVps34, its regulatory partner p150, and Beclin 1 (Atg6 in yeast).
Atg12 conjugated to Atg5 by Atg7 and Atg10. Atg12-5 then facilitates transfer of Atg8 from Atg3 to PE. Atg8-Pe then form scaffold from membrane expansion

13. Know how to compare the two processes
KNOW THE DIFFERENCES OF THE TWO PATHWAYS , CLEARLY LISTED ON YOUR HANDOUTS

14. Catabolic disease
Having said what is normal, describe how and why things can get abnormal
2) Define “proteinopathy” and give examples of neurodegenerative disease

15. Proteostasis: protein homeostasis Protein Quality Protein synthesis
Transcription &
Translation
Protein Quality
Control
Chaperones & Degradation
Neurodegeneration = death of neurones
Proteins are dynamic: they unfold/misfold and refold with the help of chaperones
Proteostasis: collection of biological processes that control the concentration of proteins present in a cell including protein concentration, conformation, binding interactions and subcellular location often through transcriptional and translational changes in reaction to environmental changes. CHANGES IN CELLULAR PROTEOSTATIS RESPONSIBLE FOR DISPOSITION OF ABERRANT PROTEIN ACCUMULATION INSIDE CELLS.
Accumulation in inclusion bodies may be due to alterations in protein synthesis such as increased protein production or defect-prone protein predisposed to more misfolding and everything this entails ir abnormal protein protein ineraction, accumulation and aggregation such as in Familial Parkinson disease in which you have multiplication of the a synuclein gene. Another scenario is that there is mutation in the genes encoding for disease associate proteins could lead to production of proteins that have high propensity to aggregate without actually changing its aount
Abnormal protein protein interactions
Inclusion bodies
Familial PD-α synuclein
Mutant Protein
Abnormal protein -protein interactions
Inclusion bodies

16. Quality control in cells
CHAPERONES:
Quality control in cells
Chaperones are proteins that assist in the normal protein formation. They help in the non-covalent folding or unfolding and the assembly or disassembly of un- or mis-folded proteins to prevent abnormal protein interactions and dysfunctional proteins.
A macromolecule is usually flexible and dynamic. It can change its shape in response to changes in its environment or other factors; each possible shape is called a conformation, and a transition between them is called a conformational change. A macromolecular conformational change may be induced by many factors such as a change in temperature, pH, voltage, ion concentration, phosphorylation, or the binding of a ligand. Proteins are dynamic and can change shape in response to changes in the environment such ie pH, ion concentration etc. Chaperones try to resist such change by promoting the normal folding of the protein. In the diseased state protein changes conformational state exhibit as abberant abberant interactions, aggregation etc. When insoluble firbils aggregate we have neurodegenerative disease

17. Progression to proteinopathy
Normal protein
Chaperone
Conformational change
Misfolded proteins
UPS/Autophagy
-aging &PD result in defective functioning of UPS
-Parkin-E3 ligase
-UCH-L1 –DUB
-VCP
Undegraded proteins =
protein aggregates
Familial disease
Oligomers β-pleated sheet forming PROTOFIBRILS
Parkin E3- component of E3 Ub ligase involved in degradation of toxic proteins
UCH-L1 found in nerve cells throughout the body
Neurodegenerative disease can be linked to defects in protein turnover as seen by the abberant deposition of ubiquitinated and disease-associate proteins in proteinacious inclusions
Proteinopathies: any disease caused (especially neurodegenerative) cause by a malformed protein. Aggregation and deposition of proteins in inclusion bodies inside or outside neurones which will cause neurodegeneration. Conformational change in proteins means different binding sites are exposed leading to protein protein interactions and aggregation. Neurodegenerative diseases are proteinopathies. Familial disease: Ubiquitinated proteins found in inclusion bodies due to inability of the UPS in neurodegenerative disease. Found that in PD and aging proteolytic activity of the proteosome is decreased- Mutation in Parkin- E3 ligase and UCH-L1-DUB, VCP
Fibrils β-pleated sheet forming
INSOLUBLE FIBRILS
Intra- Lewy body
Α synuclein
Intra-Neurofibrilliary tangle - tau
Extra-plaque

18. State what is a proteinopathy, relate to NDD and give examples
Proteinopathy: any disease (especially neurodegenerative) cause by a malformed protein.
Aggregation and deposition of proteins in inclusion bodies inside or outside neurones will cause neurodegeneration (Parkinson’s, Alzheimer’s. Frontotemporal dementia, Amylotropic lateral sclerosis).
Ubiquitinated protein were found in inclusion bodies such as Lewy bodies, tangles, plaques etc
MAP tau: microtubule associated protein tau
Lewy bodies: abnormal aggregates of proteins that develop inside nerve cells, a Lewy body is composed of α synuclein associated with other proteins such as Ubq
A-synuclein, tau: aggregate prone proteins strongly dependent on UPS and autophagy for their clearance
Parkinsons Disease
Lewy body
Intraneural
α synuclein
Alzheimers Disease
Intraneural
MAP tau
Extraneural
Amyloid β plaque

19. Neurodegenerative diseases
Commonalities
Differences
Proteinopathies
Conformational changes in proteins,accumulation,
aberrant protein protein interactions , aggregation
neurodegeneration
Neurones
Proteins
Proteinopathies associated with the accumulation of intracellular toxic proteins in neurones. Primarily caused by aggregates in the following structures:
cytosol, e.g. Parkinson’s and Huntingtons
nucleus, e.g. Spinocerebellar ataxia type 1 (SCA1)
endoplasmic reticulum (ER)
extracellularly excreted proteins, amyloid-β in Alzheimer disease
Associated with the accumulation of intracellular toxic proteins
Intracellular mechanisms
[edit]Protein degradation pathways
Parkinson’s disease and Huntington’s disease are both late-onset and associated with the accumulation of intracellular toxic proteins. Diseases caused by the aggregation of proteins are known as proteinopathies, and they are primarily caused by aggregates in the following structues:[1]
cytosol, e.g. Parkinson's & Huntington's
nucleus, e.g. Spinocerebellar ataxia type 1 (SCA1)
endoplasmic reticulum (ER), (as seen with neuroserpin mutations that cause familial encephalopathy with neuroserpin inclusion bodies)
extracellularly excreted proteins, amyloid-β in Alzheimer’s disease
There are two main avenues eukaryotic cells use to remove troublesome proteins or organelles:
ubiquitin–proteasome: protein ubiquitin along with enzymes is key for the degradation of many proteins that cause proteinopathies including polyQ expansions and alpha-synucleins. Research indicates proteasome enzymes may not be able to correctly cleave these irregular proteins which could possibly result in a more toxic species. This is the primary route cells use to degrade proteins.[1]
Decreased proteasome activity is consistent with models in which intracellular protein aggregates form. It is still unknown whether or not these aggregates are a cause or a result of neurodegeneration.[1]
autophagy–lysosome pathways: a form of programmed cell death (PCD), this becomes the favorable route when a protein is aggregate-prone meaning it is a poor proteasome substrate. This can be split into two forms of autophagy: macroautophagy and chaperone-mediated autophagy (CMA).[1]
macroautophagy is involved with nutrient recycling of macromolecules under conditions of starvation, certain apoptotic pathways, and if absent, leads to the formation of ubiquinated inclusions. Experiments in mice with neuronally confined macroautophagy-gene knockouts develop intraneuronal aggregates leading to neurodegeneration.[1]
chaperone-mediated autophagy defects may also lead to neurodegeneration. Research has shown that mutant proteins bind to the CMA-pathway receptors on lysosomal membrane and in doing so block their own degradation as well as the degradation of other substrates.[1]

20. TDP43 cellular degeneration
VCP – Valosin-containing protein Ub
p97/VCP
VCP involved in both pathways
Segregates ubiquitinated substrates of multiprotein complexes for subsequent recycling and degradadtion . Ubiquitously expressed protein belonging to AAA-ATPase family of proteins. VCP delivers substrates to the 26S proteosome thereby facilitating degradation of many proteins. If defective= protein accumulation.
IBMPFD: inclusion body myopathy (IBM) associated with Paget’s disease of bone and frontotemporal dementia (FTD) :each disease can exist individually but also on IBMPFD. Linked with IBMPFD (Inclusion body myopathy associated with Paget’s diase of bone and frontotemporal dementia) and ALS. The affected individual exhibits progressive muscle, bone and brain degenaration. Progressive autosomal dominant disorder caused by mutations in p97/VCP.
VCP binds to specific cofactors NPl14/Ufd1
AAA-ATPase: ATPases associated with multiple cellular activities .
VCP facilitates Intracellular protein degradation via UPS and pathology
VCP member of AAA-ATPase family and participates in multiple cellular processes (see handout) one of which is to facilitate protein degradation of aggregate prone protein
Facilitates delivery of ubiquitinated substrates to proteosome
ERAD: Endoplasmic reticulum associated protein degradation: eliminates misfolded/unassembled proteins from ER. VCP acts to extract substrates from ER membrane and lumen into cytoplasm for degradation by 26S. A mutant VCP will therefore lead to accumulation of ubiquitinated inclusions and protein aggregates
VCP triages ubiquitinated protein to both UPS and autophagy. VCP defect equals accumulation of ubiquitinated substrates that would otherwise be eliminated by UPS or autophagy
CFTR= cystic fibrosis transmembrane conductance regulator
The VCP basically triages degradation destined proteins to the UPSor autophagy system. It interacts with interacts with ubiquitinated proteins mediating their transport to destruction. It is also instrumental for the ERAD pathway as it transports misfolded proteins such as ΔF 508 CFTR from ER to the UPS in the cytosol for desturction. VCP dysfunction in turn leads to impaired protein degradation giving TDP-43(cellular protein) pathology (due to autiphagy impairment) and cellular degeneration. VCP id rewuired for autophagosome lysosome function: decrease in autophagic flux results in p62(Atg8) accumulation. Autophagosomes are being formed but there degrdadation is defective.
IBMPFD is a multisystem degenerative disorder of which ubiquitinated inclusions are a key pathological feature in muscle brain bone and is caused by mutations in chaperone p97/VCP. Pathology due to impaired degradation of proteins by UPS
Autophagy
TDP43 cellular degeneration
UPS
Cytocol/ERAD ic CFTR

21. Brief summary/Conclusion+
Clinical Relevance:
Delineating the molecular mechanisms underlying neurodegeneration and identifying the culprits to proteinopathy could help direct appropriate treatment