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Protein modifications 1Protein acetylation

Jeffrey S. Smith

Types of protein modifications

•Phosphorylation•Acetylation•Methylation•Ubiquitylation•ADP-ribosylation•Sumoylation•Neddylation•Glycosylation•Nitrosylation

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Acetylation

Acetylgroup

•Covalently attached to amino groups by acetyltransferases.

O

CH3 C N

H

C……...

•Acetyl CoA is used as the acetyl donor.

Acetyl-Coenzyme A (acetyl CoA)O

CH3 C S CoA

thioester bond

TerminalSulfhydryl

•Acetyl CoA carries an activated acetyl group, just as ATPcarries an activated phosphoryl group.

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How is acetyl-CoA produced (in yeast)?glucose

pyruvate

Glyoxylatecycle

glycolysis EtOH

Acetaldehyde

AcetateAcetyl-CoAFatty acids

TCAcycle

Acs1 (mito)Acs2 (nucl/cyto)

β-oxidation

acetylation

PDH

othersources

PDH = mitochondrial pyruvate dehydrogenaseACS = acetyl-CoA synthetase

fermentationPDC

ADH

ALDH

Nuclear/cytoplasmic Acs2p produces the acetyl-CoA thatis utilized by histone acetyltransferases in yeast

α-Acs2 DNA stain

Inactivation of Acs2p quicklyreduces histone acetylation

Geneexpression

Takahashi et al. 2006

(~95% nuclear)

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Catalyzed by N-acetyltransferases (NATs)N

CAc α-amino group of N-terminal

amino acids

N-terminal acetylation of proteins

•Non-reversible.•Occurs in all three kingdoms of life.•One of the most common protein modifications in eukaryotes.•Occurs on 80-90% of mammalian cytosolic proteins.•~50% of yeast proteins.•Rare in prokaryotes and Archea.•Co-translational in eukaryotes, post-translational in prokaryotes.•Activity of NATs on ε-amino groups (lysines) is not ruled out.

The three main types of yeast N-terminal acetyltransferases

Type NatA NatB NatC

Catalytic subunit

Auxiliary subunits

Substrates

Ard1p

Nat1pOthers?

Nat3p

Mdm20p

Mak3p

Mak10pMak31p

Ser-Ala-Gly-Thr-Cys-Val-

Met-Glu-Met-Asp-Met-Asn-Met-Met-

Met-Ile-Met-Leu-Met-Trp-Met-Phe-

Polevoda and Sherman, 2003

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Functional consequences of N-acetylation

•Originally thought to protect nascent protein chain from degradation, but this hypothesis is now out of favor.

•NatA-dependent acetylation of the largest origin recognitioncomplex (ORC) subunit, Orc1p, is required for transcriptionalsilencing at telomeres. (also multiple pleiotropic effects)

•NatB-dependent acetylation of tropomyosin is requiredfor interaction with actin.

•NatC-dependent acetylation of the L-A double-strandedRNA virus gag protein is a required for viral particle assembly.

Post-translational protein acetylation

Histone acetyltransferases (HATs)

Carried out by:

Factor acetyltransferases (FATs)same enzymes performboth activities

Most characterization of enzymes performed with histone substrates

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HISTONESare

highly conserved,small, basic proteins

H1

H2B

H2A

H3

H4

helix

variable

conserved

Linker histone

Core histones

N

The basic structure of ALL core histones is the same:• 1 long hydrophobic alpha-helix, bordered by• 2 short hydrophobic alpha helices that form pairs• H2A - H2B and H3 - H4 which interact.

References: Moudrianakis et al. PNAS 88, 10138 (1991); PNAS 90, 10489 (1993); PNAS 92, 11170 (1995)

The Histone Fold

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146 bp DNA

Flexible histone tails not structured.

mammals

S. cerevisiae

mammals

S. cerevisiae

H4

H3

Conservation of the histone N-terminal tails

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Acetylation of lysine neutralizes the positive charge

Acetylation of Nε-lysine

Gel scans: alfalfa histones (Waterborg, 90’s)

electrode:

electrode:

H4 102 aa

H3 135 aa

H3H4

+

_

Gel system: SDS

Separation by: size

H3

H4

+

_

102 aa

135 aa

+

+

++ +

+ +

AU (Acetic Acid – Urea)1M acid, pH ~3 in 5-8 M urea

size + charge

Separation of histones by electrophoresis

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3H-acetate labeling of yeast histone H3

Coomassiefluorogram

Acetylgroups

012345

AU gel

(-)

(+)

A-type HATs

B-type HATs

Historically, HATs were classified based on their suspected cellular origins and functions

•Cytoplasmic•Transport of histones to the nucleus•Histone deposition onto newly replicated DNA

•Nuclear•Transcription-related

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Identification of HAT1 as a cytoplasmic B-Type HAT

(Parthun et al. 1996)

AB

C D Recombinant Hat1

(Kleff et al. 1995)

HAT assay on various sources of histones

HAT1acetylatesH4

Recombinant Hat1 loses specificity

complex

Edman microsequencing

Chromatin assembly factor preferentially utilizes H4acetylated on K5 and K12.

However, hat1∆ mutants do not have any obvious phenotypes.•TPE defect in combination with H3 tail mutations.

Michael Grunstein

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Links between histone acetylation and transcription

•Allfrey et al. (1964) Identified first correlation between histoneacetylation and RNA synthesis.

•Hebbes et al. (1988) First chromatin IP experiment directlyshowed >15-fold enrichment of a transcriptionally active gene(α D-globin) relative to inactive ovalbumin gene.(using α-acetylated H4 antibody)

•Areas of acetylation correlated with DNAse-sensitive regions

Braunstein et al. (1993) Transcriptionally silent loci in yeastcorrelated with hypoacetylated histones.

Hypoacetylation (Blue) *Strong internucleosomal interactions: histone tails constrain wrapping of DNA onnucleosome surface

Hyperacetylation (Yellow) *Weak internucleosomal interactions: histone tails do not constrain DNA, which isaccessible to transcription factors

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Previous studies have established that the sedimentation coefficients of the unfolded, intermediately folded, andhigher-order folded conformational states of a 208-12 nucleosomal array are 29S, 40S, and 55S, respectively

These data indicate that an average of 12 acetates per histone octamer was sufficient tosubstantially disrupt folding of all nucleosomal arrays in the sample. They further show that highlyacetylated nucleosomal arrays were incapable of folding beyond an intermediate state.

Disruption of Higher-Order Folding by Core Histone Acetylation Dramatically Enhances Transcription of Nucleosomal Arrays

by RNA Polymerase III intermediately and higher-order folded conformational states

Tse et al (1998) Mol Cell Biol, 18:4629-4638

Sed coeff

+++ +Acetylation

Identification of the first A-type HAT

David Allis’ lab(Brownell et al. 1996)

Tetrahymenanuclear extracts

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p55 from Tetrahymena is a homolog of yeast Gcn5

*Gcn5 is a transcriptional coactivator

Recombinant Gcn5 and Esa1 are unable to acetylate nucleosomes

Grant et al. 1997

Ni2+Ni2+

•Purification of the first HAT complexes (from yeast)

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Histone acetyltransferases as large, multisubunit complexes

200

11697.4

66

45

31

21.5

**

***

*

SLIK SAGA

Tra1

Spt7

Ada3Spt8 TAF90

TAF68TAF60

(Ada5)

TAF25

TAF17

Spt20Ada1

Other Spt Ada TAF

Gcn5Ada2Spt3

Spt7

Rtg2

Tra1

Ada3TAF90TAF68TAF60

(Ada5)

TAF25

TAF17

Spt20Ada1

OtherSptAdaTAF

Gcn5Ada2 Spt3

200

11697

66

45

31

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Patrick Grant1.8 Md

C D A B

A

E D A B

GNATs

MYST

p300/CBP

Conserved domain structure

R/Q-X-X-G-X-G/A

acetyl-CoA recognitionand binding

Multiple HAT family members

YBF2/***

*

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Carrozza et al. 2003

GNAT-related acetyltransferase complexes

Different complexesoften share proteincomponents such asTra1.

Carrozza et al. 2003

Tra1 is shared with some MYST family complexes

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Carrozza et al. 2003

Certain HAT subunits are involved in targeting

Tra1

Structure and Catalytic Mechanism

GNATs vs. MYST

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Gcn5 Hat1

Tet p55

From Roth et al. 2001

Catalysis requires formation of an ordered ternary complex

1st 2nd

Peptide binding requires Gcn5/acetyl-CoA complex

red=negative

blue=positive

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*

*acetyl group not in structure

Glu173 acts as a general base catalyst to deprotonate K14

Roth et al. 2001

Mechanism of acetylation by Gcn5

Lysine ε-amino group is protonated at physiological pH.(non-reactive)

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Structure of Esa1 (MYST) HAT vs. Gcn5

Central core of catalytic domain (dark blue, domain A) is similar in the two types of HATs

Yan et al. 2002

Proposed mechanism of acetylation by Esa1

Ping-pong mechanism

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Small molecule HAT inhibitors

IC50 = 0.5 µM on PCAF and p300 (GNATs)

...

Natural:•Anacardic acid (from cashew nut shell oil)•Curcumin (ancient spice from the ginger family)•Garcinol (from the Kokum Butter tree)

The only known HAT activator is CTPB (a derivative of anacardic acid). Specific for p300 and does not work on PCAF.

Curcumin

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Kim et al. (2006) Substrate and Functional Diversity of Lysine Acetylation Revealed by a Proteomics Survey. Mol. Cell. 23:607-18.

Proteomics screen for acetylatedproteins from HeLa cells

Mitochondria(20%)

195 different proteins

Functional consequences of acetylation on non-histone substrates

Others: tubulin, TAFI68, NF-κB, Ku70, ISWI, Notch1, PPAR-γ

So far, none reported in yeast