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BMIS 310 > Leary > Flashcards

Leary Flashcards

1
Q

Does our genome encode for adequate functional diversity at the protein level?

A

No

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2
Q

Why is there a need to increase the functional diversity of our proteome?

A

Maintain homeostasis
Adapt to different conditions
To have fast biological responses that are specific
To be able to work in a crowded cellular environment

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3
Q

What potential mechanisms exist to increase the functional diversity of our proteome

A

Genetic variation: SNPs that alter protein activity (Good and bad)
Proteolytic processing: Functional activation
Splice variation: Generation of unique protein isoforms
Post-translational modification

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4
Q

How can a protein’s function be regulated?

A

Alter amount
Change its localization
Change its structure

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5
Q

T/F all vertebrates have an immune system

A

True

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6
Q

3 basics of adaptive immunity

A

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

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7
Q

Antibodies are part of which arm of adaptive immunity? What cells make them?

A

humoral arm

B-cells

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8
Q

What is an antigen

A

any molecule that elicits an immune response

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9
Q

What are antibodies? Structure?

A

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

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10
Q

Functions of antibodies? Which region is responsible for which function?

A

Two distinct functions
Bind specifically to antigen: variable region (V)
Destroy the antigen once bound: Constant region (C)

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11
Q

What interactions are there between antigen and antigen-binding sites

A

non-covalent, need lots to bind tightly

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12
Q

T/f a single antigen can only elicit the formation of one antibody

A

False
A single antigen can elicit the formation of several different antibodies
* may recognize the same portion or different portion(s) of the antigen

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13
Q

Why is flexibility at the hinger and the V-C junction important

A

Enables binding of both arms of the antibody to antigenic sites

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14
Q

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?

A

Negative
Spleen

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15
Q

How do we generate antibody diversity

A

Combinatorial diversification: shuffling a deck

Junctional diversification

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16
Q

Are antibody-antigen interactions reversible?

A

Yes
Reversible until we have enough time to create antibodies with enough complementarity

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17
Q

What does the strength of an antibody-antigen interaction depend upon?

A

Affinity and avidity

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18
Q

What is affinity and avidity

A

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

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19
Q

Why is affinity maturation integral to an effective immune response?

A

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

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20
Q

Which domains drive affinity maturation

A

Variable domains of H & L chains drive maturation

Which contains 3 discrete regions that are hypervariable

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21
Q

Why are the hypervariable regions required?

A

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

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22
Q

What do T-cells do

A

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

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23
Q

Describe affinity and avidity levels at initial immune response and end response

A

Initially low affinity, high avidity: antibody-antigen interaction is relatively weak

After maturation: increases the affinity

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24
Q

What is SCID?

A

Autoimmune disorder
Absence of T-cells and lack B-cell function

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25
Q

Why do we care about antibodies? which diseases is it relevant to?

A

Critical to adaptive immunity
Very relevant to several human diseases: SCID, autoimmune, Mitochondrial

In the lab: it s a powerful analytical reagent

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26
Q

How do you generate an antibody

A

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

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27
Q

What if the antigen is really small? can you still make an antibody?

A

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

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28
Q

Why do we use the keyhole limpet hemocyanin for small antigens

A

We use it because it’s evolutionary distant from us which is what we want. We don’t want to use similar organisms

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29
Q

How do polyclonal antibodies have an advantage over monoclonal antibodies?

A

Polyclonal antibodies are able to recognize multiple antigenic determinant

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30
Q

disadvantages of polyclonal antibodies

A

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

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31
Q

Why are monoclonal antibodies hard to generate? What did Milstein and Kohler discover?

A

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

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32
Q

What is the way that kohler and Milstein discovered? How efficient

A

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

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33
Q

How do we get rid of the plasma myeloma cells in the hybridoma mixture?

A

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

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34
Q

How do we separated the different hybridomas in the mixture

A

Positive selection
ELISA
flow cytometry etc…

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35
Q

Polyclonal vs monoclonal antibodies

A
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36
Q

Steps of western blotting

A

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

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37
Q

Why transfer proteins onto nitrocellulose rather than simply working directly with the original gel

A

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

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38
Q

Why use a secondary antibody

A

Allows one to detect each and every primary antibody
Amplification of primary antibody signal

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39
Q

What do you learn from Western blotting

A

Abundance
Molecular weight
PTMS
Processing/maturation of pre-protein

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40
Q

Why do we want to study protein folding

A

Folding determines functions

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41
Q

What are some of the cellular implications associated with protein folding and targeting?

A

Misfolding diseases which can then be targeted in the future

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42
Q

How does a protein transition from its primary amino acid sequence to a tertiary structure

A

Covalent bonds hold the primary structure together
Non-covalent and covalent bonds hold the secondary structure together
Proteins are capable of self assembly

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43
Q

What is conformation

A

Defined, three-dimensional shape of the polypeptide chain

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44
Q

What is the native conformation

A

Each protein folds into its most stable energy state

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45
Q

What are the two major models of protein folding?

A

Hierarchical folding process

Folding process driven by hydrophobic collapse

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46
Q

Do all proteins fold spontaneously to their native state?

A

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

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47
Q

Describe the hierarchical folding process

A

Local secondary structure forms initially

Supersecondary structures then form via long-range interactions

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48
Q

Describe the folding process driven by “hydrophobic collapse”

A

Spontaneous collapse into a compact state
Amino acids which that are hydrophobic collapse into the center and it shifts into the correct state

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49
Q

What is the advantage of having regions of high and low stabilities in a protein

A

The partially stable nature of proteins is used to survive and adapt to different environments

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50
Q

What mechanisms exist to promote refolding of undesired, semi-stable intermediates

A

Chaperones

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51
Q

What do chaperones do

A

Interact with unfolded or partially folded polypeptides to promote folding or provide microenvironments in which folding can occur

Two classes

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52
Q

What is the primary factor that distinguishes a prokaryotic cell from a eukaryotic one

A

It’s compartmentalized

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53
Q

Where is the bulk of protein synthesized relative to where it fulfills its function

A

Synthesized on ribosomes in the cytoplasm. So they need signal tags to let them know where they need to go.

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54
Q

What are the strategies to achieve dual localization of a protein

A

One gene
Two gene

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55
Q

What is the one gene strategy to achieve dual localization

A

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

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56
Q

What is the two gene strategy to achieve dual localization

A

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.

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57
Q

How can one protein go to different places

A

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

58
Q

What is the significance of glycosylation

A

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

59
Q

How much of the human proteins are glycosylated

A

> 50% of human proteins are glycosylated

60
Q

O-linked vs N-linked glycan structures

A

O- linked is very simple, very small, single sugar addition up to maybe 4

N-linked is insanely complicated

61
Q

N-linked vs O-linked
(Co/post translational, further modification, dolichol pyrophsophate required?, Soluble and membrane bound?)

A
62
Q

Describe N-linked glycosylation

A

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

63
Q

How does N-linked glycosylation work

A

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

64
Q

What does oligosaccharide transferase do

A

Scans proteins for Asn-X-Ser/Thr

In the lumen

Once it is scanned then it is transferred

65
Q

What does the removal of glucose make? Why is it added just to be removed

A

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)

66
Q

Which molecule recognizes if the protein is not properly folded

A

Calnexin

67
Q

What are the two things that can happen to the high-mannose

A

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

68
Q

Why are the sugars important for proteins

A

Important for conferring the desired activity and function

Important for protecting the proteins as well

69
Q

how many consensus motifs does N-linked glycosylation require

A

Single

70
Q

Describe the path of glycans to the plasma membrane

A

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

71
Q

Addition of what targets a glycoprotein to the lysosome

A

Mannose-6-phosphate

72
Q

How many different glycans are there

A

7000 unique strucutres
500 enzymes for glycosylation
minor population of processing glycosidases
majority are glycosyltransferases

73
Q

With so many competing glycosyltransferases how is glycan diversity faithfully generated?

A

Very ordered and sequential process

It is like an assembly line
Differentially regulated enzyme expression and/or localization which controls substrate availability

74
Q

What are the 3 different golgi compartments? Why are they important

A

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

75
Q

What is local redox state mainly determined by?

A

The most significant oxidizing and reducing reactions

Minor influences from transmembrane movement of electrons/protons

76
Q

What is the redox potention of the subcellular environment controlled by

A

Two primary regulatory systems

Glutathione (GSH): Glutathione disulphide (GSSG)
GSH:GSSG

Reduced/oxidized thioredoxin

77
Q

Describe the thioredoxin mechanism

A

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

78
Q

Describe the GSH/GSSG mechanism

A

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

79
Q

Do both redox couples utilize thiol-disulphide redox

A

Yes

80
Q

Are redox couples redundant

A

no

81
Q

How are the two thioredoxin and GSH/GSSG similar and different

A

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

82
Q

What is the primary determinant of redox balance

A

GSH
Thioredoxin cannot compensate

83
Q

Describe the biochemical approach in quantifying redox state? What is the challenge? Can you overcome?

A

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

84
Q

What is the cell biological approach of quantifying redox state? What is needed?

A

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

85
Q

How can we modify roGFP to examine subcellular differences in redox potential?

A

Specific subcellular location provides a direct real-time sensor with which to quantify its redox state

86
Q

How did they get roGFP more sensitive

A

attach it to Grx which is the primary target of glutathione

87
Q

What is the second oxidizing environment within the cell

A

Mitochondria

88
Q

Describe the oxidative protein folding

A

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

89
Q

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

A
  1. Spontaneous collapse of reduced cysteinal sulfur and improper formation of disulphide bonds
  2. spontaneous proper disulphide bond formation
  3. PDI pathway
90
Q

Describe the 3rd pathway which uses PDIs

A

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

91
Q

T/F PDIs are both chaperones and isomerases

A

True

92
Q

Describe the oxidative protein folding in the IMF

A

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

93
Q

What do SDS and b-mercaptoethanol do in SDS

A

SDS breaks down weak non-covalent bonds
b-mercaptoethanol breaks down disulfide bonds (reducing agent)

94
Q

What did the development and application of the slug conjugation protein show about hydroxylation

A

Dysregulated in several cancers

95
Q

What does nitrosylation do

A

can lead to both activity or inhibition

96
Q

What are some advanced class redox regulation

A

S-nitrosylation, hydroxylation, glutathionylation

97
Q

which form of glutathione is dying to react with an oxidized species

A

Reduced

98
Q

Post-translational modification of how many cysteines allows for precise functional tuning of a transcription factor

A

singular

99
Q

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?

A

The forms of the protein are not stable, we need to be able to stabilize them to study them

Stabilizing agent
Specific detection tool

100
Q

Two classes of cysteinyl redox regulation classes

A

Canonical: S-thiolation, dethiolation

Advanced: Nitrosylation, glutathionylation, hydroxyation

101
Q

T/F Proteolysis is a death sentence

A

False

102
Q

What are the two classes of targeted proteins for degradation

A

misfolded

Native, folded, critical regulatory proteins

103
Q

How does proteolytic machinery distinguish between mature, misfolded proteins, and nascent immature polypeptides?

A

Nascent proteins are physically protected

104
Q

How do proteins determine their lifetimes

A

Carry signals that determine their lifetime

1 minute to motnhs

105
Q

Even though there are multiple pathways for protein degradation, all cargo goes where?

A

Lysosome or proteasome

106
Q

What is the N-end rule

A

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

107
Q

What are the 3 enzymes needed for ubiquitination

A

E1, E2, E3

108
Q

Functions of E1, E1, E3? How specific are they?

A

E1 activating
E2 Conjugating
E3 Ligase

E2 and E3 are specific to a given protein or small subset

109
Q

What are proteasomes? Structure?

A

Large macromolecular complexes formed by 2 units, central cylinder and regulatory particles

110
Q

What is the function of regulatory particles in proteasomes

A

Receptors

111
Q

What do the fate of ubiquitinated proteins depend on

A

Ubiquitin number
Polyubiquitin configuration

112
Q

T/F ubiquitination is irreversible

A

False

113
Q

T/F the ER does not contain any proteolytic machinery

A

True

114
Q

How are misfolded proteins degraded int eh ER

A

Targeted via ubiquitination to the proteasome

115
Q

Does the mitochondria contain proteases?

A

Yes a variety

Proteases in every organelle compartment

116
Q

What is autophagy? Why do it? Where does it happen

A

Self-eating
Evolutionary conserved process in eukaryotes

Garbage disposal
Adaptive response
biosynthetic

Around the phagophore

117
Q

Which common set of ATG proteins do micro & macroautophagy share

A

ULK1 &ULK2

Core machinery

118
Q

What contributes to the progression of Parkinson’s

A

Inability to clear defective mitochondria

119
Q

What are the broadstages of autophagy

A

Phagophore biogenesis
Autophagosome formation
Autophagosome maturation (fusion with lysosome)
Breakdown of cargo
Recycling

120
Q

Describe the targeted autophagy mechanism? How does it affect Parkinson’s

A

PINK1 & PARKIN mutations cause Parkinsons

PINK1 recruits PARKIN to damaged mitochondria

PARKIN poly ubiquitinates Ub VDAC1

p62 binds poly Ub-VDAC1

121
Q

Two broad approaches for studying protein localization? What are some techniques within these broad approahces

A

Biochemical (Cell fractionation & Western Blotting)

Cell biological (Immunohistochemistry/ indirect immunofluorescence & direct fluorescence)

121
Q

antibodies may not be available for your protein of interest? What are three ways of getting them?

A

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

121
Q

What kind of environment is the cytosol

A

Highly reducing

122
Q

Describe the steps of immunohistochemistry/indirect immunofluorescence

A

The tissues are are fixed
there is a permaabilization step
Remainder is analagous to Western Blotting

123
Q

Describe direct fluorescence? What’s one advantage?

A

fusing a protein with a fluorophore allows one to determine its localization directly

High throughput

124
Q

What is the third way protein function can be regulated

A

Modify the protein structure

PTMs alter protein structure to regulate or tune function

125
Q

What is a physical method to identify protein-protein interactions? What are some challenges

A

Co-immunoprecipitation

Signal to noise
False positives
False negatives

126
Q

is co-immunoprecipitation?

A

Elute protein X then test for the presence of protein Y

127
Q

How do you identify unknown protein interactions

A

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

128
Q

When do we use proximity ligation

A

WE use it when target or unbiased physical approahces fail to capture interactions

129
Q

How do we use proximity ligation

A

We fuse a bait protein with an enzyme capable of modifying protein in proximity

130
Q

What are the two classes of enzymes

A

Biotin ligases
peroxidases

131
Q

Describe the biotin ligation mechanism? Describe the peroxidase mechanism

A

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

132
Q

Why does proximity ligation beneficial? Challenges?

A

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

133
Q

Why use a library-based method?

A

Biological responses are often very complex and highly variable

Protein-protein interactions are generally transient and weak in nature

134
Q

What does the yeast two hybrid rely on

A

Relies on the modular nature of activator proteins

135
Q

How do you get transactivation of expression

A

You only need the DNA-binding domain and activation domain

136
Q

Why fuse DNA-binding domains and activator domains

A

Fused to bait and prey proteins

137
Q

How does a yeast two hybrid work

A

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

138
Q

Advantages and disadvantages of yeast two hybrid

A

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

139
Q

What is the yeast two-hybrid using the split-ubiquitin system

A

Allows one to detect interactions between integral membrane proteins

BMIS 310 (8 decks)

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