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Bioc 212 > Midterm > Flashcards

Midterm Flashcards

1
Q

What are 3 major points about Cell Chemistry?

A
  1. Life depends on chemical reactions
    A. takes place in aqueous solution
    B. Based overwhelmingly on carbon compounds
  2. Most of the carbons present are incorporated in macromolecules
    A. Allow cells to grow and function
  3. Cell chemistry is very complex
    A. Many interlink networks of chemical reactions
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2
Q

What are the two types of chemical interactions and their properties?

A

Covalent:
- 100x stronger than non-covalent bonds
- form macromolecules
- resist being pulled apart by thermal motions
- Only broken by biologically catalyzed chemical reactions

Non-covalent:
- Allow molecules to recognize each others reversible associate

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3
Q

What is a main characteristic of chemical interactions/chemical bonds?

A

Bond strength –> is the amount of energy needed to break it.

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4
Q

Describe the chemical components of the cell.

A

99% of the total number of atoms in the cell C, H, N, O.
0.9% total number of atoms in the cell P, S, Cl, Na, Mg, K, Ca.
There a certain combinations of atoms (chemical groups) that are abundant in cells:
Methyl (-CH3)
Hydroxyl (-OH)
Carboxyl (-COOH)
Carbonyl (-C=O)
Phosphate (-PO3^-2)
Sulfhydryl (-SH)
Amino (-NH2)

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5
Q

How are cell compounds formed?

A
  • Carbon atoms can form four covalent bonds with other atoms –> they have a high ability to form macromolecules
  • C-C stable bonds forms chains and rings -> generate large and complex molecules
  • Carbon compounds made by cells -> organic compounds
  • A few categories of molecules give rise to all extraordinary richness of form and function
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6
Q

Describe organic compounds.

A
  • Carbon based (around 30 carbons)
  • Found in free solution
  • Common example: hydrogen, oxygen, nitrogen
  • Compounds in the cell are chemically related and classified in 4 major families of compounds
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7
Q

What are the uses of organic compounds?

A

1) Monomer subunits to construct Polymeric Macromolecules
2) Energy sources –> broken down and transformed into other small molecules (used in metabolic pathways)
3) Many have both functions (subunits and energy sources)
4) Organic molecules are synthesized of or broken down into the same set of simple compounds

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8
Q

Describe the transition of organic compounds to macromolecule.

A

Organic compounds - small organic building blocks of the cell
Macromolecule - large organic molecules of the cell
Sugar –> Polysaccharides, Glycogen, Starch (in plants)
Fatty Acids –> fats and membrane lipids
Amino Acids –> proteins
Nucleotides –> Nucleic Acids

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9
Q

What are macromolecules?

A
  • Most abundant carbon containing molecules
  • Principal building and functional blocks of cells
  • Made by covalently linked organic molecules (monomers) into chains
  • Proteins are an important macromolecule - versatile and perform thousands of functions, enzymes which catalyze formation and breaking of covalent bonds
  • Nucleotide –> nucleic acid –> DNA and RNA
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10
Q

Describe the assembly of macromolecules.

A
  • They assembly in a sequence, not randomly, subunits are added in a precise order
  • Covalent bonds allow rotation and give flexibility, which allows for several conformations
  • Non-covalent bonds allow assembly of macromolecules but constrain the shape to one conformation - but they still allow them to interact with one another
  • What happens in the cell brings order
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11
Q

What are the two types of reactions in cells?

A

Anabolic:
Two monomers put together through covalent bonding - need to provide energy to make bonds.
Condensation - releases H20
Energetically unfavourable

Catabolic:
Break bonds and makes monomers - releases energy when bonds are broken.
Hydrolysis - takes in H20
Energy favourable

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12
Q

What does the second law of thermodynamics state? How is it possible?

A
  • In any isolated system the degree of disorder always increases
  • The most probable arrangement is the most disorder (if there is no energy in system)
  • Quantified by ENTROPY (S) - the greater the disorder the greater the entropy is
  • Systems will change spontaneously towards arrangements with higher S
  • This is possible if you consider cells as non-isolated system. It has an environment. Interactions inside the cell have order and also release heat to its environment. Warms up the environment. It heats the liquid as it moves faster which creates disorder.
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13
Q

Where does cell heat come from?

A
  • Cell heat comes from food
  • It is release unless it needs to create more order in the cell
  • The food molecules feed heat (catabolic pathway) and makes many building blocks for biosynthesis (and releases heat in the process) Then goes through anabolic pathways which makes molecules that form the cell.
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14
Q

What is the first law of thermodynamics?

A

The energy can be converted from one form to another but not created or destroyed.

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15
Q

In biological systems, how is energy stores and managed?

A
  • It is stored and managed within chemical bonds
  • Enthalpy (H) –> energy that can be released from chemical bonds
  • A negative enthalpy change (Hf-Hi) spontaneously favourable reaction
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16
Q

What is Gibbs free energy?

A

Putting it all together
DeltaG = DeltaH -TDeltaS
When DeltaG is 0, it is energetically favourable.

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17
Q

What is the purpose of an Enzyme?

A

Enzymes catalyze the reaction by lowering the activation energy required for a reaction to take place. It then takes less time for the reactants to reach the required activation energy, thereby speeding up the reaction.

Enzymes speed reactions but cannot force energetically unfavourable reactions to occur. (cannot go uphill)

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18
Q

Explain what this statement means “reactions in cells are coupled.”

A
  • The energy in one reaction is used in another reaction (they drive each other)
  • Need carrier molecules to take energy and favour anabolic reactions - main energy carrier in our cell is ATP
  • In order for the cell to work, DeltaG = (-)
  • In order for work - it depends on the concentration of components that we have - this is the concept of equilibrium in the cell
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19
Q

Explain the concept of Equilibrium in the cell.

A
  • Suppose there is a reaction which is Y–> X
  • In this example, the formation of X is favourable. It is negative. And formation of Y is unfavourable, it is positive. There will always be some X converting to Y.
  • Suppose we start with equal number of X and Y.
  • For each individual molecule, conversion of X to Y will happen less often then Y to X. Therefore the ratio of X to Y molecules will increase.
  • Eventually, there will be a large enough excess of X over Y to just compensate for the slow rate of X –> Y. Eventually equilibrium will be attained.
  • At equilibrium, the number of Y molecules being converted to X molecules (vice versa) is the same. There is no net change in the ratio of Y to X.
  • This is known as the Standard free energy and it depends on the components and delta G.
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20
Q

Describe an example of a reaction driven by ATP hydrolysis.

A

Reaction: A-O + B-H —> A-B

Step 1: In the ACTIVATION step, ATP transfers a phosphate, P, to A-OH, to produce a high energy intermediate.

Step 2: in the CONDENSATION step, the activated intermediate reacts with B-H to form the product A-B, a reaction accompanied by the release of inorganic phosphate.

Then there is the net result.
A-OH + B-H + ATP –> A-B+ ADP + P

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21
Q

What is Acetyl CoA?

A
  • It is a molecule that provides energy
  • See it a bit less often
  • High energy Thioester bond - used to transfer energy through reactions
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22
Q

Describe oxidation and reduction involved in electron transfer.

A
  • Oxidation in the cells -> it is a catalyzed reaction and refers to more than the addition of oxygen (addition of oxygen rarely happens), removal of electrons from the atoms. Partially + charge.
  • Reduction -> Addition of electrons to an atom. Partially - charge. If a molecule picks an e- it usually also pics an H. This is called hydrogenation.

When reduces it is methane.
When it oxidizes it is carbon dioxide.
In a cell, reduction and hydrogenation is the same.

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23
Q

What is an example of hydrogenation? When does it equal reduction?

A

A + e- + H+ –> AH
Hydrogenation = Reduction if the number of C-H bonds increases, the molecule is then reduced.

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24
Q

What is NADH and NADPH?

A

They are both electron carriers
When two reactions take place, energy is taken by one reaction to another, you then have ATP carriers.
When there is oxidation in a reaction, molecules that carry electrons and hydrogen is NADH and NADPH.

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25
Q

Pair up the following activated carries with the group that they carry in a high-energy linkage.
1. ATP
2. NADH, NADPH, FADH2
3. Acetyl CoA
4. Carboxylated biotin
5. S-Adenosylmethionine
6. Uridine diphosphate glucose

A
  1. Phosphate
  2. Electrons and hydrogens
  3. Acetyl group
  4. Carboxyl group
  5. Methyl group
  6. Glucose
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26
Q

Which is the most abundant macromolecule in the cell?

A

Proteins!

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27
Q

Describe the diversity between protein structures. Why is this significant?

A
  • Diverse structures have multiple functions
  • Proteins are cell building blocks, they provide shape and structure.
  • They undertake most functions; enzymes catalyze cell chemical reactions, membrane proteins form communication channels, transport of cargo and mechanical forces.
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28
Q

How do proteins acquire their function?

A

Proteins acquire function by folding into a 3-dimensional conformation
Folding provides physical stability and functional surfaces
The sequence of amino acids of a protein determines its structure, function, and localization

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29
Q

What are proteins made from?

A

Proteins are polymers that are made of 20 different amino acids (monomer)
A polymer is a peptide chain - made up by amino acids.
In the centre there is a carbon that makes 4 covalent bonds to amino, carbon, carboxyl (acidic) and to X.

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30
Q

What is the general formula for an amino acid?

A

The general formula for an imo acid is a Carbon in the centre, attached to a R (side-chain group), COOH (carboxyl group), NH2 (amino group), and a H.

R is commonly one of 20 different side chains. At pH 7 both the amino and carboxyl groups are ionized.

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31
Q

What are the three characteristics that side chains (R) have?

A
  1. Hydrophobic, polar, or charge (acidic or basic)
  2. Small or large
  3. Covalently linked into polypeptides
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32
Q

Name the 5 polar amino acids and describe their structure.

A
  1. Asparagine (Asn or N) - Has one CH2 and an Amide chain.
  2. Glutamine (Gln or Q) - has two CH2 and an Amide chain.
  3. Serine (Ser or S) - has one CH2 and an OH.
  4. Threonine (Thr or T) - has one CH, one CH3, and one OH.
  5. Tyrosine (Tyr or Y) - Has a CH2 and benzene ring, then OH.
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33
Q

What pH are amino acids charged at?

A

They are at pH 7.

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34
Q

What are the 3 basic amino acids and describe their name and structure. (positively charged)

A
  1. Lysine (Lys or K) - 4 x CH2 group and NH3 +
  2. Arginine (Arg or R) - 3 x CH2 group, NH, C attached to NH2 and NH2 +
  3. Histidine (His or H) - CH2 attached to C attached to HN, HC, NH+, CH (the nitrogens have a weak affinity for an H and are only partly positive at neutral pH)
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35
Q

What are the 2 Acidic amino acids and describe their name and structure. (negatively charged)

A
  1. Aspartate (Asp or D) - CH2 attached to C double bond O and single bond O-
  2. Glutamate (Glu or E) - 2x CH2 attached to C double bond O and single bond O-
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36
Q

What does the R group allow for between basic and acidic amino acids?

A

Electrostatic/ionic interactions

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37
Q

What are the 10 hydrophobic nonpolar amino acids? What are their name and structure?

A
  1. Alanine (Ala or A) - CH3
  2. Glycine (Gly or G) - H
  3. Valine (Val or V) - CH2 attached to 2 x CH3
  4. Leucine (Leu or L) - CH2 - CH to 2 x CH3
  5. Isoleucine (IIe or I) - CH - CH3 and CH2 - CH3
  6. Proline (Pro or P) - N attached to CH2 - CH2 - CH2 attached to C.
  7. Phenylalanine (Phe or F) - CH2 - benzene ring
  8. Methionine (Met or M) - CH2 - CH2 - S - CH3
  9. Tryptophan (Trp or W) - CH2 - square triangle NH to benzene ring
  10. Cysteine (Cys or C) - CH2 - SH
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38
Q

What are two characteristics about hydrophobic nonpolar amino acids?

A
  • They interact through hydrophobic interactions (exclusion of water molecules)
  • Disulphide bonds can form between two cysteine side chains in proteins
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39
Q

How are amino acids joined together?

A
  • Amino acids are joined together by amide linkage, called a peptide bond.
  • Peptide bonds in the backbone of the polypeptide are uncharged but polar.
  • Charge and hydrophobicity of a polypeptide is determined by the side chains.
  • Both side chains and backbone can form non-covalent contacts with other AA.
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40
Q

When two amino acids are linked together, what do the two single bonds allow for?

A

They allow for rapid rotation, so that long chains of amino acids are very flexible.

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41
Q

Polypeptide Backbone

A
  • The peptide bond is planar and cannot rotate
  • Rotation around the bonds to the central carbon is possible
  • The polypeptide backbone has limited freedom of rotation
  • Some rotation angles between amino acids (residues) in a polypeptide are preferred
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42
Q

What is a non-covalent bond? What are four examples?

A
  • Interactions between residues of a polypeptide stabilize structure
    1. Hydrogen bonds
    2. Van der Waals interactions (transient dipoles between all atoms)
    3. Ionic bonds
    4. Hydrophobic interactions (exclusion of water) - molecules of water can’t interact with it so then it has to interact with other water which creates a cage and inside is the hydrophobic molecule (amino acid), it takes a lot of water to isolate the hydrophobic residue so it is better to have them together in one big cage.
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43
Q

Describe the covalent interaction between cysteines:

A
  • Disulfide Bonds
  • Secretory proteins often have covalent disulfide bonds between cysteine side chains
    - extracellular proteins, inside secretory organelles
    - disulfides reinforce structure
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44
Q

What proteins usually do not have disulfide bonds?

A
  • Cytosol, nucleus, mitochondria
  • Usually not in proteins in the cytoplasms because it is a reductant vitamin and not able to make bonds
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45
Q

How many organization levels contribute to protein structure?

A

4

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46
Q

What is a key feature about Primary and Secondary structure?

A

Primary - Linear amino acid sequence

Secondary - Local conformation patterns

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47
Q

Describe the Alpha-Helix of the Secondary Structure:

A
  • Alpha- helix: single polypeptide chain twisted around on itself
  • Backbone is coiled
  • Hydrogen bonds between C=O and N-H formed every 4 peptide bonds in each turn of helix backbone
  • Side chains point outwards
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48
Q

Describe the Beta-Sheets of the Primary Structure:

A
  • Beta sheets: neighboring segments of the polypeptide backbone
  • backbone is extended almost straight
  • Several strands pack sideways into a Beta sheet
  • hydrogen bonds between the backbone strands
  • Side chains alternate sides
  • Very rigid structure
  • There are two types; Antiparallel and Parallel
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49
Q

Describe the Tertiary structure. How is it formed? What are some characteristics?

A
  • Complete three-dimensional arrangement of the polypeptide
  • Secondary structure elements are packed against each other to form the tertiary structure
  • Hydrophobic contacts between secondary elements
  • Long- range contacts between residues that are far apart in the primary sequence
  • Held together by Hydrogen bonds, Ionic bonds, and hydrophobic interactions.
  • the loops that are formed have no regular secondary structure and can be flexible.
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50
Q

Describe the Quaternary structure. How is it formed? What are some characteristics?

A
  • The assembly of multiple polypeptides (subunits) into a final protein
  • Interactions between subunits are very stable
  • Dimer: two polypeptide subunits
  • Trimer, Tetramer, 5-mer, 6-mer, etc.
  • Oligomer: many subunits
51
Q

What are 4 different ways/representations to visualize proteins?

A
  1. Polypeptide backbone
  2. Ribbon diagram (polypeptide backbone only)
  3. Stick diagram (includes AA R chain)
  4. Space-filling model (with mass of atoms)
52
Q

What is a Domain?

A
  • A domain is an independently folded unit within a protein
  • Proteins can one one or multiple domains
  • Different domains in a protein often have different functions
53
Q

What are Domain Combinations?

A
  • Some conserved domains are found in many different proteins, in combination with other domains
  • These “modular” domains often form reversible, specific, non-covalent contacts with other molecules
    - other proteins (different from quaternary structure)
    - lipid, carbohydrates, RNA, DNA, other cofactors
54
Q

What is a length of a Polypeptide?

A
  • Most human polypeptides are 100 to 800 amino acids long, or form 12 kDA to 90 kDA molecular weight
  • Domains are usually 50 to 200 amino acids long
  • Long proteins have multiple domains
55
Q

Why are protein interactions often transient?

A
  • This means they form and break apart quickly
  • This is because thermal motion means all molecules are constantly moving and tumblings and colliding
  • There is a binding equilibria
56
Q

What is sequence similarity and when is it useful/what does it indicate?

A
  • Sequences of different polypeptides can be compared with each other, to align identical (same AA) and similar amino acids (same properties)
  • Sequence similarity (homology) indicates evolutionary conservation
  • Homology suggests a common structure or function
  • If polypeptides do not have sequence similarity, they are considered divergent.
57
Q

What is a protein family?

A
  • A family is a set of proteins or domains which have homologous sequences and structures
  • They often have related functions
  • An organism can have several proteins from the same family
  • proteins from a family can be found in different organisms
58
Q

Which amino acids can form the following interactions?
1. Hydrophobic interactions
2. Hydrogen bonds
3. Van der Waals interactions
4. Ionic bonds
5. Disulphide bonds

A
  1. A, I, L, V, P, F, W, M, (Y) (C, G)
  2. D, E, R, K, H, N, Q, S, T, Y, (W)
  3. all
  4. D, E, R, K, H
  5. C
59
Q

What interaction can polypeptide backbone form?

A

Hydrogen bonds, Van der Waals.

60
Q

What is Post-Translational Modification (PTM)? What is it important for/What are the 3 different types of modification?

A
  • Proteins can be chemically modified after translation
  • Important contribution to proteomic diversity and complexity and essential for regulation of protein function and cellular signalling
  • Different types;
    1. Cleaved into smaller proteins by peptidases
    2. Covalent modification of N-terminus (co-translational)
    3. Covalent modification of side chains: Introduce functional groups to proteins
61
Q

(PTM) What are Side chain modifications used for?

A
  • They are used for various cellular functions
  • Can change the surface or conformation of protein
  • Can create or block a binding site for other proteins
  • Many modifications are regulated and reversible
  • Modifications are fast, so useful as switches (switch to send signal but then can turn it off again)
  • determine if protein will now interact with another protein or not
62
Q

(PTM) what are the modifications mediated by? what are the main types of modifications?

A
  • the modifications are mediated by enzymes (but follow the rules of organic chemistry)
    main types:
  • Phosphorylation
  • Methylation
  • Acetylation
  • Glycosylation, Sumoylation, Ubiquitination
63
Q

PTM: Phosphorylation. What is it? What transfers and removes the phosphate?

A
  • Major regulatory mechanism
  • Phosphorylation on hydroxyl groups (S, T, Y)
  • Adding a phosphoryl group changes the charge and size
  • Kinases (enzyme) transfer phosphates from ATP, specific for side chain and the surrounding peptide sequence.
  • Phosphatase’s removes phosphate.
64
Q

PTM: Phosphorylation of S, T, Y.
What is the Kinase families vs the Phosphatase families?
(super important for signalling)

A

Kinase:
Ser/Thr kinases
Tyr
Dual specificity (Ser/Thr and Tyr)

Phosphatase families:
Ser/Thr phosphatases
protein Tyr phosphatases
Dal specificity (Ser/Thr and Tyr)

65
Q

Describe Phosphopeptide Binding. What is an example?

A
  • This is when specialized domains bind p-SER, p-Thr or p-Tyr
  • phosphorylation is required for binding
  • Surrounding polypeptide sequence also contributes

Example: WD40 domain of Cdc4 with the Sic1 CPD peptide with pThr.

66
Q

Describe the Acetylation of Lysine

A
  • Acetylation of Lys amine changes the polarity (isopeptide bond)
  • Lysine (K) acetyltransferases (KATs) and deacetylases (KDACs), originally histone acetyltransferases. They recognize specific sequences. (they are enzymes that aid in this process)
  • Signalling and metabolic effects
  • Increase in size
  • Change in charge (example - histone acetylation = active transcription)
67
Q

Describe the methylation of Lysine and Arginine.

A
  • Methylation of Arginine involves the addition of 1 or 2 methyl groups to the guanidino group.
  • Methylarginines -> N-Methylarginine is an inhibitor of nitric oxide synthase. Chemically, it is a methyl derivative of the amino acid arginine.
  • Add size to K and R
  • Lysine can be mono, di, or trimethylated
  • Two enzymes that aid are: Lysine methyltransferases (KMT) and lysine demethylases (KDMs)
  • Remember that the two amino acids that can be methylated are lysine and ariginine.
68
Q

How does PTM binding work?

A
  • Like phosphorylation, acetylation & methylation provides new binding sites for proteins
  • Specific domains bind Ac-Lys, Me-Lys, Me-Arg and surrounding sequences
69
Q

What does the folded structure of a protein depend on? Where are the polar side chains found?

A
  • The folded structure depends on hydrophobic interactions
  • Polar side chains are found on the outer surface
70
Q

What is the “native state” of the protein?

A
  • It is the completely folded conformation of a protein
  • Most stable conformation of the protein
  • Structure is stabilized by hydrophobic contacts (exclusion of water)
  • Some domains also require a ligand partner to be stable - cofactor (Haem, steroid, etc) or another protein subunit.
  • Native state can be in equilibrium with near-native state folding intermediates
71
Q

Rank/Describe the interactions importance for folding?
1. Hydrophobic interactions
2. hydrogen bonds
3. Van der Waals interactions
4. ionic bonds
5. disulfide bonds

A
  1. Many, strong
  2. Many, moderate
  3. Many, weak
  4. Few, very strong
  5. Few, covalent (very strong)
  • Hydrogen bonds stabilize secondary structures and are involved in peptide bonds
  • Hydrophobic interactions between secondary structures form the tertiary structures –> involve the side chains
72
Q

What is the “native structure” determine by? What is its state?

A
  • It is determined by the primary sequence of AA
  • It is the state of minimal energy: folding is thermodynamically favoured (negative deltaG free energy
73
Q

Describe the Protein Folding Process:

A
  • Folding is a complex process (different free energy conformations)
  • Unfolded (denatured) domains have extended conformations with no secondary or tertiary structure
  • Folding proceeds through intermediates that have increasing structure, to the native state
74
Q

What is a folding intermediate? What is there a risk of?

A
  • Have some secondary structure, but tertiary structure incomplete
  • Some hydrophobic side chains are exposed instead of buried
  • More of the polypeptide is flexible and disorder
  • Risk of aggregation:
    - hydrophobic regions prefer to be in contact with others
    - interaction between different unfolded proteins leads to insolubility
75
Q

What is the result of a misfolded protein?

A
  • Immediately after protein synthesis
  • required ligand not available
  • genetic mutation –> misfolded of proteins –> Disease (sickle cell anemia, cystic fibrosis)
  • Harmful environmental conditions (heat) lead to unfolding and misfolding of properly folded proteins
  • Aging: decrease efficiency of the protein quality control mechanisms, lose of protein homeostasis, harmful aggregates of misfolded proteins (amyloid), Neurodegeneration (Alzheimer, Parkinson, ALS, dementia)
76
Q

What can be the result of a mutation?

A
  • Can lead to changes in polypeptide sequences
  • Amino acid substitution, insertion or deletion, or premature stop
  • can disrupt the folding or function of protein
77
Q

What are Allelic variations? What can they sometimes cause?

A
  • Allelic variation describes the presence or number of different allele forms at a particular locus (locus or loci = place) on a chromosome
  • Can sometimes cause genetic disease
78
Q

What does Protein Homeostasis (Proteostasis) refer to?

A

It refers to an extensive network of components that acts to maintain proteins in the correct concentration, conformation, and subcellular location, to cooperatively achieve the stability and functional features of the proteome.

79
Q

What are at the centre of the protein quality control network?

A
  • Chaperones are at the centre
  • They are very important, if you removed them from the genome cells and they would die etc.
80
Q

Describe what chaperones are: (+ examples)

A
  • Molecular chaperones assist folding and prevent aggregation, without being part of the native state
  • Chaperons often recognize exposed hydrophobic regions of folding intermediates
  • Chaperons are consecutively expressed and essential under non-stress conditions
  • Many chaperones are Heat Shock Proteins (HSP, eg. Hsp70 is the 70 kDa HSP), highly expressed after stress.
81
Q

Describe Heat Shock Response (HSR) and Unfolded protein response (UPR): (chaperones)

A
  • Cells respond to stress that causes protein misfolding by increasing the expression of chaperones and other specialized proteins
  • HSR: cytosolic and nuclear proteins, protects against cell death
  • UPR: ER proteins, can promote cell death if stress is too severe
  • Cells tailor the expression of chaperones (HSPs) to the level of unfolded and misfolded proteins.
82
Q

What are constitutive chaperons?

A
  • They assist protein folding
  • They are proteins that facilitate the folding of others
  • Hold or stabilize hydrophobic residues
  • Universal mechanism of protein homeostasis
83
Q

How is stress response/heat shock response activated?

A

It is activated by unfolded cytosolic proteins
- Heat stress
- Oxidative damage
- proteasome inhibition

84
Q

is the transcription of heat shock proteins up- regulated or down-regulated? What about other proteins?

A
  • Transcription of heat shock proteins is up regulated
  • Other proteins are down-regulated
85
Q

Are many heat shock proteins chaperones?

A

Yes

86
Q

Does the response of HSP’s continue after the stress is removed?

A

Yes it still continues, this is to help the cells recover.

87
Q

What is HSF1 and what is its function?

A
  • HSF1 is a transcription factor that mediates the heat shock response
  • It activates the transcription of HSPs
  • Inactive HSF1 is monomeric, active HSF1 is a trimer
  • Active HSF1 recognizes HSE (heat shock element) promoters

HEAT SHOCK TRANSCRIPTION FACTOR

88
Q

What is the regulation of HSF? (5 steps)

A
  1. Monomeric HSF1 is folded, but mimics unfolded protein and is bound by Hsp90
  2. After heat shock, unfolded proteins compete with HSF1 for Hsp90 binding
  3. Free HSF1 trimerizes and activates transcription
  4. Chaperones including Hsp90 are expressed and help fold or degrade unfolded proteins
  5. HSF1 is down-regulated by binding of excess Hsp90 to the monomer
89
Q

What is the difference in function between ATP-dependent and ATP-independent chaperones?

A

ATP-dependent chaperones actively promote folding
- Substrate binding and release are regulated by ATPase cycles

ATP-independent chaperones prevent aggregation and can catalyze some folding steps

90
Q

What are places where there is cooperation between chaperones?

A

Cytosol & Endoplasmic Reticulum

91
Q

What are the names of the 3 ATP-dependent chaperones?

A
  1. Hsp70 Family
  2. Hsp90 Family
  3. Chaperonins (Hsp60)
92
Q

What does HSP60 function like?

A

E. Coli GroEL

93
Q

Which is the only chaperone that isnt always expressed?

A

HSP70

94
Q

Which 3 chaperones are induced by heat shock response?

A
  1. HSP70
  2. HSP90
  3. HSP60
95
Q

Which 2 chaperones are induced by ER Unfolded protein response?

A

BiP and GRP94

96
Q

What are some characteristics that describe the HSP70 Family?

A
  • HSP70 chaperones are 70 kDA monomers
  • The ATPase domain controls the substrate-binding domain
  • ATP-bound - no substrate peptide binding
  • ADP- bound - the substrate binding domain is closed tightly on the peptide
  • Binds short hydrophobic sequences
97
Q

Describe how HSP70 function helps co-chaperones?

A
  • Co-chaperones are proteins which contact chaperones to regulate their activity
  • Some can bind to polypeptide substrates themselves, and are both chaperones and co-chaperons
  • DNAJ (HSP40) family promotes HSP70 substrate binding
  • Nucleotide Exchange Factors (NEFs) promote substrate release
98
Q

Explain the HSP70 Functional cycle:

A

1) Hsp40-mediated delivery of substrate to ATP-bound Hsp70

2) Hydrolysis of ATP to ADP mediated by Hsp40 results in closing of the alpha-helical lid and tight binding of substrate by Hsp70

3) NEF catalyzes exchange of ADP for ATP

4) Opening of the alpha-helical lid, induced by ATP binding, results in substrate release

5) Released substrate either folds to native state (N)

99
Q

What chaperones are found in the Cytosol for HSP70?

A
  • HSC70 and HSP70
100
Q

What chaperones are found in the Cytosol for HSP90?

A
  • HSP90 alpha and beta
101
Q

What chaperones are found in the Cytosol for Chaperonin (HSP60)?

A

TRiC

102
Q

What chaperones are found in the ER for HSP70?

A

BiP

103
Q

What chaperones are found in the ER for HSP90?

A

GRP94

104
Q

What are some key characteristics about DNAJ Co-chaperones?

A
  • DNAJs regulate HSP70 function
  • Many DNAJs - at least 53 genes in human cells
  • All have a conserved J domain (bind transiently to HSP70, activate it to hydrolyze ATP and bind polypeptide, do not bind substrate)
  • Other domains determine their specific biological function
105
Q

Are substrate-binding DNAJs the most highly conserved?

A

Yes

106
Q

What are the 3 domains of DNAJ? / characteristics

A
  1. J domain
  2. Substrate binding
  3. Dimerization
  • Homodimers: 2 subunits of 40-50 kDa (originally identified as HSP40)
  • Bind short hydrophobic sequences
  • Transfer substrate to HSP70 during ATP hydrolysis
  • Some DNAJs bind substrate through specific domains and act as ATP-independent chaperones
  • Some DNAJs do not bind substrate
    - Specific domains attach DNAJ to a protein complex or intracellular membrane
    - these DNAJs recruit HSP70s to the complex or membrane
107
Q

What is the function of NEFs? (HSP70 Nuclear Exchange Factors)

A
  • Nucleotide Exchange Factors (NEF) remove ADP from HSP70 and allow ATP to bind
  • NEF binding opens up HSP70 ATPase domain and weakens interactions with nucleotide
  • ATP binds when NEF dissociates
  • ATP-bound HSP70 to releases polypeptide
  • There are several NEF families in humans
108
Q

How does HSP70 Help Folding?

A
  • HSP70 binds hydrophobic regions of folding intermediates and prevents incorrect contacts from forming
  • Release of polypeptide from HSP70 provides chances for it to fold
  • Balance between DNAJs and NEFs support an optimal rate of HSP70 binding and release
  • Substrate-binding DNAJs may provide additional assistance
  • Can form multi-chaperone complex with HSP90
109
Q

Describe the HSP90 Family

A
  • HSP90 chaperones are homodimers, with 2 identical subunits joined at the C-termini - human Hsp90: 2 x 90 kDA = 180 kDa
  • Dimer can open and close, like a nutcracker
  • ATP controls opening and closing of the dimer
  • Co-chaperone p23 stabilizes closed form
  • Thought to bind polypeptides at late stages of folding
  • Binds to hydrophobic and polar surfaces –> stabilizes intermediate folded states
  • Substrate is bound along the sides of the subunits
  • Different substrates can bind to different sites on the sides - unlike HSP70s and chaperonins
110
Q

What are the 3 steps of the HSP90 Functional Cycle?

A
  1. Substrate is bound weakly in the open nucleotide-free state
  2. ATP binding allows dimer to close and bind substrate tightly
  3. ATP hydrolysis to ADP compacts the dimer and releases the substrate
111
Q

Describe the HSP70-HSP90 Cochaperone system:

A
  • Cystolic HSP70 and HSP90 forms a multi-chaperone system
  • Cooperate to assist substrates
  • Substrate is releases from HSP70 and bound by HSP90 in coordination
  • HOP assists complex formation
  • HSP70 dissociates when HSP90 binds ATP
  • Many co-chaperones regulate HSP70 and HSP90 (co chaperones sometimes act on substrates, provide flexibility, folding and non-folding functions)
112
Q

What is EEVD motif? describe it in terms of HSP70 and HSP90.

A
  • One “hotspot” for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s.
  • Cytosolic HSP70 and HSP90 are not homologous, but have similar C- terminal sequence motifs
  • HSP70: PTIEEVD-COO-
  • HSP90: MEEVD-COO-
  • TPR domains recognize these EEVD motifs
  • Can be specific for HSC70, HSP90 or both
  • In the class example, the cochaperone is HOP and it binds them together and brings both families of chaperones together, it can do this cause it has a TPR that recognized EEVD motifs.
113
Q

Describe the TPR domain co-chaperone:

A
  • TPR domains are adaptors to HSP70 and HSP90
  • TPR co-chaperones often have other domains which interact with substrate directly
114
Q

What is HOP?

A
  • It is a co chaperone
  • It has domains which bind to HSP70 and HSP90 specifically
115
Q

What is FKBP52?

A
  • It has an HSP90-binding TPR domain, PPIase domains - peptidyl-prolyl isomerase: chaperone specific to prolines
116
Q

What is CHIP?

A
  • Binds either to HSP70 or HSP90 and has a ubiquitin ligase domain that helps degrade proteins
  • Everyone chaperone has different domains
117
Q

What does HSP90 chaperones have to do with signalling?

A
  • Many HSP90 substrates are signal transduction proteins
  • Kinases, receptors, transcription factors
  • Many also require HSC70
  • Except: HSP90 binds kinases without needing HSC70
118
Q

What happens if you have a mutation in signalling proteins? What are drug targets for cancer treatment?

A
  • Mutations in signalling proteins are causes of cancer
  • HSP90 and HSC70 are drug targets for cancer treatment
119
Q

Explain the example of a mutation: v-src

A
  • If it was normal it would be c-src (cellular): normal kinase involved in signalling cell growth
  • if it was mutated, v-src (viral): mutant that causes cancer
  1. v-src expressed in fibroblast (epithelial) cells causes them to become cancerous
  2. Treat cells with Hsp90 inhibitor
  3. Hsp90 cannot chaperone v-src anymore
  4. Cells revert from cancer to normal growth
  • A side effect is the accumulation of misfolded proteins, activate HSF1 - become very weird loop- expressing more HSF proteins (side effect)
120
Q

Describe Chaperonins (HSP60 family)

A
  • chaperonins are large oligomeric complexes, with a typical double-ring structure
  • E coli. GroEL: 2 rings x 7 identical subunits x 60 kDA = 840 kDA
  • E coli. GroES cap co-chaperone: 7 subunits x 10 kDA= 70 kDA
  • Homologs of human mitochondrial Hsp60 and Hsp10
121
Q

What is GroEL Cavity?

A
  • GroEL, a tetradecameric bacterial chaperonin, is one of the most studied chaperonins, but the role of the internal cavity in the refolding process is still unclear. It has been suggested that rather than simply isolating proteins while they refold, the GroEL cavity actively promotes protein folding
  • Rings are identical and work in alternating cycles
  • there is a down position and a up position
  • In the down position ( there is no nucleotide) there are subunits around the ring bind to hydrophobic polypeptide
  • in the up position (ATP-bound) there is conformational change. The subunits bind to GroES cap instead of substrate, a large cavity with a polar surface is formed, substrate is released inside cavity: enclosed but no longer bound.
122
Q

Describe the Subunits of the GroEL?

A
  • Each GroEL subunit has an ATPase domain and a substrate-binding domain
  • The ATPase domain is the interface with the opposite ring (upside down)
  • Movement of the substrate binding domain is controlled by the ATPase in both rings
123
Q

What is GroEL and GroES? How do the the substate-binding domains bind?

A
  • GroEL-ES is a molecular chaperone complex that helps other proteins fold correctly in the cell. Each GroEL complex consists of two back-to-back rings, each composed of seven subunits. GroES is a heptameric co-chaperonin that caps the GroEL after the substrate is encapsulated, concurrently with the ATP hydrolysis.
  • Substrate-binding domain either binds substrate (down, no nucleotide) or GroES (up, ATP-bound)

Bioc 212 (11 decks)

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