Leary Flashcards
Does our genome encode for adequate functional diversity at the protein level?
No
Why is there a need to increase the functional diversity of our proteome?
Maintain homeostasis
Adapt to different conditions
To have fast biological responses that are specific
To be able to work in a crowded cellular environment
What potential mechanisms exist to increase the functional diversity of our proteome
Genetic variation: SNPs that alter protein activity (Good and bad)
Proteolytic processing: Functional activation
Splice variation: Generation of unique protein isoforms
Post-translational modification
How can a protein’s function be regulated?
Alter amount
Change its localization
Change its structure
T/F all vertebrates have an immune system
True
3 basics of adaptive immunity
Specificity: Distinguishes between self and non-self, discerns between small differences in “non-self”
Memory: Takes time for adaptive immune response to build a response
Immunity is brought about by a variety of leukocytes: Generated in bone marrow, T-cells in the thymus and B-cells in the spleen
Involves cell-mediated and humoral systems: 2 complementary systems
Responds to antigen
Antibodies are part of which arm of adaptive immunity? What cells make them?
humoral arm
B-cells
What is an antigen
any molecule that elicits an immune response
What are antibodies? Structure?
Y-shaped proteins that bind very tightly to their targets
Two light and two heavy chains that are identical
Antigen-binding sites are identical
Linked via non-covalent and covalent interactions
Functions of antibodies? Which region is responsible for which function?
Two distinct functions
Bind specifically to antigen: variable region (V)
Destroy the antigen once bound: Constant region (C)
What interactions are there between antigen and antigen-binding sites
non-covalent, need lots to bind tightly
T/f a single antigen can only elicit the formation of one antibody
False
A single antigen can elicit the formation of several different antibodies
* may recognize the same portion or different portion(s) of the antigen
Why is flexibility at the hinger and the V-C junction important
Enables binding of both arms of the antibody to antigenic sites
Do we have enough physical space in our genome to code for all the different regions of an antibody? Where do we generate antibody diversity?
Negative
Spleen
How do we generate antibody diversity
Combinatorial diversification: shuffling a deck
Junctional diversification
Are antibody-antigen interactions reversible?
Yes
Reversible until we have enough time to create antibodies with enough complementarity
What does the strength of an antibody-antigen interaction depend upon?
Affinity and avidity
What is affinity and avidity
Affinity: Strength of binding of a single copy an an antigenic determinant to a single antigen binding site
Avidity: Total binding strength of a multivalent antibody with a multivalent antigen
Why is affinity maturation integral to an effective immune response?
We are maturing and increasing the affinity of an immunoglobulin for the antigen
We are taking something that has enough chemical complementarity to bind to the antigen and maturing it to have a perfect fit
Which domains drive affinity maturation
Variable domains of H & L chains drive maturation
Which contains 3 discrete regions that are hypervariable
Why are the hypervariable regions required?
Not enough genomic space for necessary antibody diversity
Gene duplication combined with VDJ recombination provides for generation of millions of distinct antibodies
Hypermutation of these regions allows for affinity amturation of antibodies
What do T-cells do
Help with affinity maturation
Naive antibody repertoire: ensure at least one B-cell in the circulation to produce an antibody with reasonable affinity
T-cells help with somatic hypermutation: ~1 mutation per variable region per cell division
Describe affinity and avidity levels at initial immune response and end response
Initially low affinity, high avidity: antibody-antigen interaction is relatively weak
After maturation: increases the affinity
What is SCID?
Autoimmune disorder
Absence of T-cells and lack B-cell function
Why do we care about antibodies? which diseases is it relevant to?
Critical to adaptive immunity
Very relevant to several human diseases: SCID, autoimmune, Mitochondrial
In the lab: it s a powerful analytical reagent
How do you generate an antibody
First pick an antigen: computer algorithms
Synthesize and purify the antigen
Immunologically challenge an animal: Rabbit is commonly used
We get a unique response from each animal
The serum against an antigen will yield at least B cells which recognize the antigenic determinant
What if the antigen is really small? can you still make an antibody?
If it is less than 5 kDa our immune system does not mount a response
Yes we use a limpet hemocyanin is arguably the most widely used carrier
Why do we use the keyhole limpet hemocyanin for small antigens
We use it because it’s evolutionary distant from us which is what we want. We don’t want to use similar organisms
How do polyclonal antibodies have an advantage over monoclonal antibodies?
Polyclonal antibodies are able to recognize multiple antigenic determinant
disadvantages of polyclonal antibodies
Each antiserum is different even if raised in genetically identical animals
Antiserum is produced in limited volumes, using the same reagent in a long series of experiments may not be possible
Antiserum may include minor populations of antibodies that give unexpected cross reactions
Why are monoclonal antibodies hard to generate? What did Milstein and Kohler discover?
Each antibody forming B-cell is specialized for the synthesis and secretion of only one antibody
and B-lymphocytes cannot be cultured in isolation so you can’t amplify it
Discovered a way to propagate B-lymphocytes in culture by fusing them with cells derived from mouse myeloma cells to proliferate
What is the way that kohler and Milstein discovered? How efficient
We challenge an animal with an antigen then we harvest the spleen
The B-cells harvested are mortal
We fuse the plasma cells and myeloma that forces them to become hybridoma
This process is not perfectly efficient we will also have normal B cells and myeloma cells in the hybridoma mixture (with time the B-cells would fall off, but the myeloma/plasma cells wouldnt divide)
Negative selection
Positive selection
How do we get rid of the plasma myeloma cells in the hybridoma mixture?
Negative selection
Force all the cells to divide by salvage pathway. Plasma cells cant do it so they eventually die.
Use HAT selection: hypoxanthine, aminopterin, thymidine
How do we separated the different hybridomas in the mixture
Positive selection
ELISA
flow cytometry etc…
Polyclonal vs monoclonal antibodies
Steps of western blotting
Sample preparation: Denature with SDS and reducing agents
Gel electrophoresis: Separate by size
Transfer: transfer to membrane
Blocking: block the unbound binding sites of the membrane to remove background
Detection: Secondary antibody and tag
Why transfer proteins onto nitrocellulose rather than simply working directly with the original gel
A membrane is easier to handle and manipulate without it breaking
Low concentrations are more easily detected, because they are concentrate on the surface
staining and destaining is faster
The blot is convenient
Why use a secondary antibody
Allows one to detect each and every primary antibody
Amplification of primary antibody signal
What do you learn from Western blotting
Abundance
Molecular weight
PTMS
Processing/maturation of pre-protein
Why do we want to study protein folding
Folding determines functions
What are some of the cellular implications associated with protein folding and targeting?
Misfolding diseases which can then be targeted in the future
How does a protein transition from its primary amino acid sequence to a tertiary structure
Covalent bonds hold the primary structure together
Non-covalent and covalent bonds hold the secondary structure together
Proteins are capable of self assembly
What is conformation
Defined, three-dimensional shape of the polypeptide chain
What is the native conformation
Each protein folds into its most stable energy state
What are the two major models of protein folding?
Hierarchical folding process
Folding process driven by hydrophobic collapse
Do all proteins fold spontaneously to their native state?
No, some proteins are too long and it would take way too long to fold to the native conformation when it has to happen in milliseconds to seconds
Describe the hierarchical folding process
Local secondary structure forms initially
Supersecondary structures then form via long-range interactions
Describe the folding process driven by “hydrophobic collapse”
Spontaneous collapse into a compact state
Amino acids which that are hydrophobic collapse into the center and it shifts into the correct state
What is the advantage of having regions of high and low stabilities in a protein
The partially stable nature of proteins is used to survive and adapt to different environments
What mechanisms exist to promote refolding of undesired, semi-stable intermediates
Chaperones
What do chaperones do
Interact with unfolded or partially folded polypeptides to promote folding or provide microenvironments in which folding can occur
Two classes
What is the primary factor that distinguishes a prokaryotic cell from a eukaryotic one
It’s compartmentalized
Where is the bulk of protein synthesized relative to where it fulfills its function
Synthesized on ribosomes in the cytoplasm. So they need signal tags to let them know where they need to go.
What are the strategies to achieve dual localization of a protein
One gene
Two gene
What is the one gene strategy to achieve dual localization
One gene would create one mRNA could be acted upon by two translating ribosomes to create two translation products. One has the tag visible and the other is buried in the folds which causes them to go to different places
What is the two gene strategy to achieve dual localization
Two genes (alternative methionine start site) creates two mRNAs. Which creates two translation products, one has the tag visible, one has it buried causing it to go to different places.
How can one protein go to different places
The tag could be visible or invisible due to folding, the tag could be blocked by a protein interaction, or there could be 2 tags and there is competition for the protein
What is the significance of glycosylation
Found in every kingdom of life
Eukarya: protein folding and quality control, targeting, cell-cell recognition
Bacteria & Archaea: Assembly and strength protein complexes, adapt to environment, virulence factor
Prevalent in bioactive natural products
Glycoengineering: Doping
How much of the human proteins are glycosylated
> 50% of human proteins are glycosylated
O-linked vs N-linked glycan structures
O- linked is very simple, very small, single sugar addition up to maybe 4
N-linked is insanely complicated
N-linked vs O-linked
(Co/post translational, further modification, dolichol pyrophsophate required?, Soluble and membrane bound?)
Describe N-linked glycosylation
Irreversible, covalent attachment of a glycan to a target protein
Derived from a 14-sugar unit
PTM exhibits heterogeneity
Variety of structural and/or functional roles
How does N-linked glycosylation work
Requires 15 Asn Linked Glycosylation genes (ALG)Built up sequentially on a dolichol phosphate backbone
First two steps are cytosolic
Flippase flips it to the ER lumen where the next 7 units are added
What does oligosaccharide transferase do
Scans proteins for Asn-X-Ser/Thr
In the lumen
Once it is scanned then it is transferred
What does the removal of glucose make? Why is it added just to be removed
Generates high mannose
It is important for protein folding and quality control. The thought is that once it is added it signifies a mature protein so it is ready to leave the ER
(don’t completely know why)
Which molecule recognizes if the protein is not properly folded
Calnexin
What are the two things that can happen to the high-mannose
Leaving the one branch alone and just have it as mannose and then diversify the other branch creating a hybrid
Or
Prune the tree down to the base and create two highly complex arms creating a complex
These bring structural stability to the proteins
Why are the sugars important for proteins
Important for conferring the desired activity and function
Important for protecting the proteins as well
how many consensus motifs does N-linked glycosylation require
Single
Describe the path of glycans to the plasma membrane
The base 14 sugar unit that was created in the ER
The glucoses are important so the OST recognizes the glycan is ready to add to the asparagine
Its then exported as high mannose to the golgi
If it is going to the plasma membrane, we either get a hybrid or complex sugars
Addition of what targets a glycoprotein to the lysosome
Mannose-6-phosphate
How many different glycans are there
7000 unique strucutres
500 enzymes for glycosylation
minor population of processing glycosidases
majority are glycosyltransferases
With so many competing glycosyltransferases how is glycan diversity faithfully generated?
Very ordered and sequential process
It is like an assembly line
Differentially regulated enzyme expression and/or localization which controls substrate availability
What are the 3 different golgi compartments? Why are they important
Cis-Golgi, Medial-golgi, Trans-golgi
Different things are available in each place
Unique environments
Glycan generation relies on appropriate expression and compartmentalization of glycosyltransferases and substrates
What is local redox state mainly determined by?
The most significant oxidizing and reducing reactions
Minor influences from transmembrane movement of electrons/protons
What is the redox potention of the subcellular environment controlled by
Two primary regulatory systems
Glutathione (GSH): Glutathione disulphide (GSSG)
GSH:GSSG
Reduced/oxidized thioredoxin
Describe the thioredoxin mechanism
Sulfurs are reduced which interacts with a substrate.
The substrate is reduced
The thioredoxin then is reduced by a NADPH dependent reaction catalyzed by TrxR
Describe the GSH/GSSG mechanism
You have 2 reduced glutathione which spontaneously gives up its proton which will reduce any oxidized substrate
To go back to its reduced state it is NADPH dependent and is catalyzed by GSHR
Do both redox couples utilize thiol-disulphide redox
Yes
Are redox couples redundant
no
How are the two thioredoxin and GSH/GSSG similar and different
Simialr in that they transfer reducing equivalents from NADPH to an oxidized substrate and they are small redox-active
Different in that they are not interchangeable, only GSH is capable of spontaneously acting as a nucleophile
GSH in not an enzyme
Thioredoxin is an enzyme
What is the primary determinant of redox balance
GSH
Thioredoxin cannot compensate
Describe the biochemical approach in quantifying redox state? What is the challenge? Can you overcome?
Measure GSH and GSSG as an endpoint assay on a spectrophotometer
You must freeze the redox state of an entire cell
You cannot quantify in its individual compartments
You can overcome by fluorescent microscopy because you can look at a living organism in real time
What is the cell biological approach of quantifying redox state? What is needed?
GFP is needed
If you excite the GFP at a specific wavelength you can make it light up
Introducing two cysteines to the structure produces a molecule dependent on the redox state
How can we modify roGFP to examine subcellular differences in redox potential?
Specific subcellular location provides a direct real-time sensor with which to quantify its redox state
How did they get roGFP more sensitive
attach it to Grx which is the primary target of glutathione
What is the second oxidizing environment within the cell
Mitochondria
Describe the oxidative protein folding
PDIs interacts with a protein that needs to be folded which causes disulphide bond shuffling
The PDI becomes oxidized which is then rereduced by Ero1
What are the 3 scenarios when a protein goes from the cytosol to the lumen of the ER? 3 possibilities before it is determined to be unsalvageable
- Spontaneous collapse of reduced cysteinal sulfur and improper formation of disulphide bonds
- spontaneous proper disulphide bond formation
- PDI pathway
Describe the 3rd pathway which uses PDIs
Reduced cysteinyl sulphurs, the PDIs engage with those and accept those protons. It gives them up to Ero1 which gives them up to form hydrogen peroxide
T/F PDIs are both chaperones and isomerases
True
Describe the oxidative protein folding in the IMF
Mia40 has oxidized sulphurs which takes the reducing equivalents from the protein so it can form the disulphide bridge which feeds it to Erv1 which feeds it to the ETC to produce water
What do SDS and b-mercaptoethanol do in SDS
SDS breaks down weak non-covalent bonds
b-mercaptoethanol breaks down disulfide bonds (reducing agent)
What did the development and application of the slug conjugation protein show about hydroxylation
Dysregulated in several cancers
What does nitrosylation do
can lead to both activity or inhibition
What are some advanced class redox regulation
S-nitrosylation, hydroxylation, glutathionylation
which form of glutathione is dying to react with an oxidized species
Reduced
Post-translational modification of how many cysteines allows for precise functional tuning of a transcription factor
singular
What is the primary challenge with respect to detecting post-translational modifications of cysteines? What tools or strategies do we need to overcome this challenge?
The forms of the protein are not stable, we need to be able to stabilize them to study them
Stabilizing agent
Specific detection tool
Two classes of cysteinyl redox regulation classes
Canonical: S-thiolation, dethiolation
Advanced: Nitrosylation, glutathionylation, hydroxyation
T/F Proteolysis is a death sentence
False
What are the two classes of targeted proteins for degradation
misfolded
Native, folded, critical regulatory proteins
How does proteolytic machinery distinguish between mature, misfolded proteins, and nascent immature polypeptides?
Nascent proteins are physically protected
How do proteins determine their lifetimes
Carry signals that determine their lifetime
1 minute to motnhs
Even though there are multiple pathways for protein degradation, all cargo goes where?
Lysosome or proteasome
What is the N-end rule
It impacts the half-life of cytosolic proteins
M, S, T, A, V, C, G & P are stabilizing. If there is something else then they will be degraded
What are the 3 enzymes needed for ubiquitination
E1, E2, E3
Functions of E1, E1, E3? How specific are they?
E1 activating
E2 Conjugating
E3 Ligase
E2 and E3 are specific to a given protein or small subset
What are proteasomes? Structure?
Large macromolecular complexes formed by 2 units, central cylinder and regulatory particles
What is the function of regulatory particles in proteasomes
Receptors
What do the fate of ubiquitinated proteins depend on
Ubiquitin number
Polyubiquitin configuration
T/F ubiquitination is irreversible
False
T/F the ER does not contain any proteolytic machinery
True
How are misfolded proteins degraded int eh ER
Targeted via ubiquitination to the proteasome
Does the mitochondria contain proteases?
Yes a variety
Proteases in every organelle compartment
What is autophagy? Why do it? Where does it happen
Self-eating
Evolutionary conserved process in eukaryotes
Garbage disposal
Adaptive response
biosynthetic
Around the phagophore
Which common set of ATG proteins do micro & macroautophagy share
ULK1 &ULK2
Core machinery
What contributes to the progression of Parkinson’s
Inability to clear defective mitochondria
What are the broadstages of autophagy
Phagophore biogenesis
Autophagosome formation
Autophagosome maturation (fusion with lysosome)
Breakdown of cargo
Recycling
Describe the targeted autophagy mechanism? How does it affect Parkinson’s
PINK1 & PARKIN mutations cause Parkinsons
PINK1 recruits PARKIN to damaged mitochondria
PARKIN poly ubiquitinates Ub VDAC1
p62 binds poly Ub-VDAC1
Two broad approaches for studying protein localization? What are some techniques within these broad approahces
Biochemical (Cell fractionation & Western Blotting)
Cell biological (Immunohistochemistry/ indirect immunofluorescence & direct fluorescence)
antibodies may not be available for your protein of interest? What are three ways of getting them?
De novo generation
A number of monoclonal antibodies bind to a variety of short synthetic peptides
Incorporate a peptide tag into cDNA and express the protein
What kind of environment is the cytosol
Highly reducing
Describe the steps of immunohistochemistry/indirect immunofluorescence
The tissues are are fixed
there is a permaabilization step
Remainder is analagous to Western Blotting
Describe direct fluorescence? What’s one advantage?
fusing a protein with a fluorophore allows one to determine its localization directly
High throughput
What is the third way protein function can be regulated
Modify the protein structure
PTMs alter protein structure to regulate or tune function
What is a physical method to identify protein-protein interactions? What are some challenges
Co-immunoprecipitation
Signal to noise
False positives
False negatives
is co-immunoprecipitation?
Elute protein X then test for the presence of protein Y
How do you identify unknown protein interactions
Co-immunoprecipitation and mass spectrometry
Immunoprecipitated proteins have to be digested into polypeptides first
then match an experimental proteolytic profile of the unknown to proteolytic profiles in databases
Then you have to consider localization, abundance, antibodies, strength of interactions and refine the data
When do we use proximity ligation
WE use it when target or unbiased physical approahces fail to capture interactions
How do we use proximity ligation
We fuse a bait protein with an enzyme capable of modifying protein in proximity
What are the two classes of enzymes
Biotin ligases
peroxidases
Describe the biotin ligation mechanism? Describe the peroxidase mechanism
Your bait protein has biotin ligase attached. When biotin is added, biotin ligases uses biotin to label surface exposed lysines. There is a radius which limits how many proteins will be labeled. up to 10 nm. Then isolate biotinylated proteins and identify by mass spectrometry
Instead of adding biotin you add biotin-phenol and hydrogen peroxide
Why does proximity ligation beneficial? Challenges?
Can capture weaker protein-protein interactions
Context-dependent interactions
Challenges:
Biotinylation must be optimized to achieved desired signal to noise
Need appropriate control bait proteins
Physical constraints may preclude biotinylation of protein partners
tag may affect protein localization
Labeling cannot be used in certain subcellular compartments
Why use a library-based method?
Biological responses are often very complex and highly variable
Protein-protein interactions are generally transient and weak in nature
What does the yeast two hybrid rely on
Relies on the modular nature of activator proteins
How do you get transactivation of expression
You only need the DNA-binding domain and activation domain
Why fuse DNA-binding domains and activator domains
Fused to bait and prey proteins
How does a yeast two hybrid work
Bait and prey genes are cloned into distinct plasmids.
Each encoding for separate markers examples LEU and TRP
They are inserted into yeast cells which cannot produce Tryptophan, Leucine, or His.
You then grow the yeast on media without tryptophan or leucine. If they grow then they have successfully taken up the plasmids.
You then grow it on a plate without histidine and if the bait and prey interact then the reporter gene allows yeast to synthesize histidine and grow
Advantages and disadvantages of yeast two hybrid
Advantages:
High throughput and capture weak protein interactions
Disadvantages:
High false positive and false negative rate
Overexpression can lead to non-specific reactions
Biased against certain classes of proteins
What is the yeast two-hybrid using the split-ubiquitin system
Allows one to detect interactions between integral membrane proteins
