C1- Protein Modification

1. N-terminus 2. Lysine

2 different types of acetylation:

1. Farnesyl (geranylgeranyl) transferase binds to CAAX box 2. Proteolysis of AA 3. Carboxy methylation

3 steps in Farnesylation and Geranylgeranylation:

basic enzymatic mechanism

A similarity of the two different acetylation mechanisms is that they both have same _______ on amino groups

Reversible (poly) ADP-ribosylation of Glu/Asp

ADP-ribosylation that is a normal physiological response to DNA damage

ADP-ribosylation

Addition of ADP-ribose to proteins

No

Are farnesylation and geranylgeranylation reversible?

Cholera toxin

Arg modification of Gs (G-protein)

Imatinib (Gleevec)

Bcr-Abl tyrosine kinase inhibitors

overactivate

Both cholera and pertussis toxins ____ adenylate cyclase

Gly X Y X= pro Y= 4-Hydroxyproline (Hyp)

Collagen contains repeats of sequence _____

Pertussis toxin

Cyc modification of Gi (G-protein)

Different enzymes, substrates and effects on proteins

Differences of 2 acetylation mechanisms:

Irreversible (mono) ADP-ribosylation

Diphtheria, Cholera and Pertussis toxins all use this mechanism

Prolyl hydroxylase

Enzyme used to convert a prolyl residue to a 4-hydroxyprolyl residue

1. poly (ADP-ribose) glycohydrolase 2. ADP-ribosyl protein lysase (removes last ADP-ribose)

Enzymes used to remove ADP-riboses

"CAAX box" C=Cys A=aliphatic amino acid X= anything

Farnesyl transferase (and geranylgeranyl transferase) recognizes ______ and forms a stable thioether bond

cysteine residues

Farnesylation and Geranylgeranylation occur at _____

Lipid modifications to proteins

Farnesylation, Geranylgeranylation, Myristoylation and Palmitoylation are examples of _______

SOS

GEF of RAS is _____

Ca2+

Gamma-carboxylation of GLA domain allows ____ binding which facilitates membrane binding

inhibitors

HAT and HDAC ____ are potential therapies

Resveritrol

HDACs- a target of ______

Diphtheria toxin

His modification of EF2

2

How many mechanisms of transfer are there for Ub from E2 to substrate/acceptor?

Negatively

Hypoxia-inducible factor (Hif) is ____ regulated by prolyl-hydroxylation

conserved Gamma carboxyglutamic acid rich (GLA)

In Gamma-carboxylation, the enzyme targets a _____ protein domain

Protein tyrosine kinase (growth factor) receptor (PTKR)

In RAS-RAF1-extracellular signal- regulated kinase (ERK) pathway, ____ is activated by autophosphorylation and allows binding to downstream factors

RAS farnesylation

In RAS-RAF1-extracellular signal- regulated kinase (ERK) pathway, _____ facilitates membrane association

RAF1/MEK/ERK protein kinase cascade

In RAS-RAF1-extracellular signal- regulated kinase (ERK) pathway, ______ causes activation of nuclear transcription factor ELK

Warfarin

Inhibition of clotting with ______

No

Is N-terminus acetylation reversible?

yes

Is Ubiquitylation reversible?

yes

Is acetylation on lysine reversible?

Yes

Is phosphorylation reversible?

Yes

Is poly ADP-ribosylation reversible?

Immune suppression

Kinases and phosphatase are important drug targets for _____

Cancer chronic myelogenous leukemia

Kinases are important drug targets for ______

common and essential

Lipid protein modifications are __ and ____ modifications

Collagen

Major extracellular triple helix protein of Connective tissue

N-terminal methionine

Majority (80%) of proteins are acetylated on their _____

clotting cascade

Many GLA-containing proteins are found in _____

RAS

Many tumors have a mutated ____ that has lost its ability hydrolyze GTP so it is always active

Ubiquitin

Most common member of small protein family

disease

Mutations in kinase/phosphatase genes are associated with _____

fatty acid

Myristoylation and Palmitoylation involve the addition of a _____

N-terminus

Myristoylation occurs at the ______

Co-translational

N-terminal methionine acetylation is a _____ modification

Thioether bond

Palmitoylation involves a ___ bond to cysteine residues

cysteine residues

Palmitoylation occurs at ______

Fk506 (Tacrolimus)

Phosphatase calcineurin inhibitor

activity, location and stability

Phosphorylation can change protein ___,___ and ___

glutamic acid, aspartic acid or C-terminus of protein

Poly ADP-ribosylation targeted to ___, ___ or ____

proteasomal degradation

Poly ubiquitylation (K48 and K11 linkage) is the usual signal for _______

rapamycin (Sirolimus)

Protein Kinase mTOR inhibitor

sumolyation

Protein ___ does not target proteins for degradation but can regulate protein function or localization

functionality

Protein modification exceedingly common to extend the _____ of amino acids

farnesylated

RAS is ____ at C-terminus

GTPase

RAS is a _____

1. Palmitoylation 2. plasma membrane 3. removal from membrane 4. Depalmitoylation 5. golgi membrane

Steps in RAS protein modification: 1. __1__ of farnesylated RAS in golgi 2. Vesicular transport to __2__ 3. Binding to factor required for __3__ 4. __4__ 5. binding to __5__

Nicotinamide adenine dinucleotide (NAD+)

Substrate for ADP-ribosylation

1. amino 2. Ubiquitin

Substrate for E3 may be 1. Protein substrate- ____ group of lysine 2. ____ - amino group of a lysine or a amino group of the N-terminal methionine

C-terminal sequence

Synthese of Ub requires removal of _____ to generate the correct Ub C-terminus

ubiquitin ligase (E3)

Transfer of Ub from charged E2 to substrate/acceptor requires ______

1. Reversible (poly) ADP-ribosylation of Glu/Asp 2. Irreversible (mono) ADP-ribosylation

Two classes of ADP-ribosylation modification:

poly proteins

Ub genes are synthesized as ____ or fusions between Ub and another protein

recycled

Ub is ____ during proteasomal degradation

1. lysine 2. isopeptide bond

Ubiquitin is attached to __1__ residues via __2__

1. Adenylation 2. Thioesterification 3. Trans-thioesterification

Ubiquitin-activating enzyme (E1) carries out three enzymatic functions: 1. __1__ of Ub 2. __2__ of E1 3. __3__ of Ubiquitin-conjugating enzyme (E2)

E1

Ubiquitin-activating enzyme =____

E2

Ubiquitin-conjugating enzyme= _____

topologies

Ubiquitylation has different _______

E1, E2 and E3

Ubiquitylation is carried out by three enzymes:

specific

Unlike poly ADP-ribosylation, monoribosylation by bacteria toxins have very ____ substrates, with specific biochemical influences on those substrates

reductase

Vitamin K is rejuvenated by ____

poly ubiquitylated proteins

When ubiquitin is the substrate for E3, it can lead to _____

Seven lysines and N-terminal methionine

___ and ___ of Ub can be ubiquitylated

Sumo

___ is another member of the ubiquitin family

Cholera

___ toxin acts in the intestinal epithelial cells

Few (1 in yeast, 2 in humans)

____ E1 enzymes in most eukaryotes

GAP

____ can stimulate GTPase activity of RAS (results in loss of effector binding)

Warfarin

____ inhibits reductase

Farnesyl pyrophosphate

____ is intermediate in cholesterol biosynthesis

Hydroxyproline (Hyp)

____ is more abundant in animals than seven "common" amino acid residues

Hydroxyproline (Hyp)

____ is necessary for proper folding and assembly of collagen

lysine acetylation

____ of p53 tumor suppressor is required for activation

chain editing

____ takes place with Ub modified proteins

Ubiquitin

____- ubiquitously expressed small 8.5kDa protein (highly conserved)

De-ubiquitylation enzymes (DUBs)

_____ complete syntheses of new Ub

C-terminus

_____ of RAS genes are divergent

lysine acetylation

_____ stabilizes cytoskeletal microtubules

amino group

acetylation of lysine occurs on its ______

Poly (ADP-ribose) polymerase (PARP)

enzyme for ADP-ribosylation

protein quality control and degradation

function of N-terminus acetylation

Histone acetylation (HATs) and deacetylation (HDACs)

lysine acetylation is used for _______

1. cysteine of E2 to Lysine of substrate 2. Thioesterification

mechanisms of transfer for Ub from E2 to substrate/acceptor: 1. Direct transfer of Ub from active site ___ of E2 to __ of substrate 2. _____ of E3 from charged E2 followed by transfer of Ub from active site cysteine of E3 to lysine of substrate

HRAS, NRAS, and KRAS

three RAS genes:

1. Collagen 2. Hypoxia (low oxygen)- inducible factor

two substrates of prolyl-hydroxylase