Nuclear Pore Complex

Free transportation for Mr <40,000 Da

1000 translocation events per sec per NPC

NPCs serve as anchorage sites for chromatin and involved in the regulation of gene expres

sion

90 nm

8 X symmetry

FG Repeat/non-structure

FG Repeat Binding Site

FG Repeat Binding Site

Nucleoporins

Nucleoporins: glycosylated & hydrophobic

• Mammals– FXFG

• Yeasts– GLFG

Most of them located symmetrically at NPC, whereas only a few are located asymmetrically.

Blocked with the hydrophobic compound hexane di

ol

Nucleoporins

• I: Structural• II: FG to interact with karyophilins

• 50% of nucleoporins• Exoport karyophilins has a high-affinity binding site

on the cytoplasmic side.

• III: tether the NPC to the membrane• IV: WD for modulation

Karyopherins: with multiple (~20) HECT repeats

ImportinExportin

HECT: E3 ubiquitin ligase domain

Multiple Sites for Receptor Recognition

Sequential Events

Ran

• GTP-Ran in Nucleus • GDP-Ran in Cytosol

Importin/substrate in cytosol

In the presence of GDP-Ran

Importin<<<<>>>>Substrate

Exportin/cargo/GTP-Ran

RanGAP &RanBP1&RanBP2

Exportin<<<<<>>>>>Cargo

Interconnectivity of Transcription and the Ubiquitin-Proteasome Sy

stemDynamic: Exchange of Complexes

Regulation of levels of transcription factors

-catenin• c-myc• Nuclear Hormone Receptors

Nuclear Hormone Receptors

•Steroid Hormone Receptors

–In cytoplasm with chaperone

•Estrogen•Androgen•Progesterone•Glucocorticoid

• Non-Steroid Hormone Receptors– In nucleus at hormone

response elements as a heterodimer with RXR

• Vitamin D• Retinoic Acid• Peroxisome Proliferato

r-Activated

Nuclear Receptor Superfamily

Nuclear Hormone Receptors

Reactive with coactivators and AF-1

Modification of Nuclear Hormone Receptors: Transcription as a

Dynamic Process• Phosphorylation

– MAPK– Cdk7 of TFIIH

• Ubiquitination• Acetylation• Methylation• Ubiquitin-Proteasome Pathway

Chromosomal Architecture

Chromatin-Modifying Enzymes

• ATP-dependent nucleosome remodelling enzymes– SW1/SNF

• Histone-modifying enzymes– Histone acetyletransfe

rase (HAT)

Coactivators for Nuclear Hormone Receptors

• P160• Histone Aceyltransferase (HAT)• Histone Arginine Methyltransferases• Nucleosome Remodeling Complex• Multisubunit Mediator Complex: bridge nuc

lear hormone receptors and the basal transcriptional machinery

Special Ubiquitinylation

Not for degradations but for molecular interactions

Mono-Ubiquitin

H3

H3Phosphorylation Me Ac

trans

Ubiquitin as a sorting SignalGH receptor Endocytosis

EGF receptor internalizationGlutamate receptor

etc

Proteasome In Eukaryotes

Threonine Proteases

Beta1, beta2, beta5 with catalytic activity.

Beta1: post-glytamyl-peptide-hydrolyzing; beta2: trypsin like; beta5:chymotrypsin like.

Beta 1, 2 & 5catalytic

Regulatory Factors for Proteasome Assembly

• Positive– Hsp90

• Negative– PAAF-1 (proteasomal

ATPase-associated factor-1)

Closed Open

Activator & Inhibitor

19S=PA700PA28PI31

PI31

Phosphorylation

19S• Base

– Rpt1-Rpt6: ATPase– Non-ATPase

• Rpn1• Rpn2

• Lid: Non-ATPase– 8 subunits– Rpn10: a hinge between th

e base and lid– Rpn11: An intrinsic Zn2+-de

pendent metallopeptidase to release ubiquitin and peptides for 20S proteasome

– Rpn4: Binds and acts as a transcriptional activator

PA28 (Proteasome Activators)

• Alpha/beta– In immune tissues– cytoplasmic

• Gamma– For trypsin-like activity

• In brain• In nucleus

Immuno-Proteasomes

• Page 43

Fool the substrates for E3

RING

HECT

Virus

E3

• RING (Really Interesting New Gene)– Two zinc atoms– No directly associated with ubiquitin

– Such as Mdm2 to ubiquitinize -arrestin– C-Cbl

RING

E3

Homologous to E6-AP C-terminal350 a.a. residuesRecognize proline rich motifs

HECT

Ubiquitin Pathway

Ubiquitin: 76 a.a. polypeptide29K48K63K

Ubiquitin

E3

• SCF (Skip-Cullin-F-box)– Phosphorylation of he

target protein often triggers ubiquitylation

• Anaphase-Promoting Complex (APC; cyclosome) contains a RING-finger protein (Apc11), an E2 and a cullin (Cdc20/Hct1).

SCF

ECSRING: Roc1

Substrate binding

HIF: Hypoxia Inducing Factor

E3: Cell Cycle Regulatory

Anaphase-promoting complex

Skp-Cul1-

F-box: with Phosphorylated substrates

Phosphorylation

Protein Folding versus Degradation

• CHIP (C-terminal Hsp70-interacting protein) contains a modified RING-finger and promotes the ubiquitylation of misfolded proteins, which are then degraded by the proteasome.

Ubiquitin like protein

Ubiquitin-Like Protein

Ubiquitin-Like Proteins: Modify substrates for different purposes than does ubiquitin itself

Protein

Lipid

Protein

Export

Crm1

Aster promoting activity

Leucine-rich Nuclear Export Signals

• Low affinity for CRM1, stimulated by RanBP2

• CRM1 –mediated translocation through the NPC is energy-independent facilitated diffusion

Sumoylation at Nuclear Pore Complex

RanBP2(Nup358)

Models

Small Ubiquitin-like Modifier (SUMO)

SUMO

•97 aa•SUMO-1, -2, -3, -4•SUMO modification occurs on the lysine in KXE

:hydrophobic

Substrates for Sumoylation

• RanGAP KXE/D• PCNA TKET• Smad4 VKYC• VKDE

Proteases for SUMO• SENP• SuPR1 & Axam2 are splice variants of

Senp2

=ubiquitin like protease 1=cysteine protease family

unique

E3 ligases

• PIAS (protein inhibitor of activated signal transducer and activator of transcription) with RING finger motif

• RanBP2• Pc2 (Polycomb Protein)

Competition between ubiquitnation and SUMO modification at lysine

• IB

Mechanisms of covalent protein attachment that have been implicated in the negative regulation of cytokine signaling

Regulation of the JAK-STAT signal transduction pathway

Domain structures of proteins that repress cytokine signal transduction

E3 ubiquitin ligase

mRNA export

NXF1-p15 (Mex67p:Mtr2p) mediate nuclear export of bulk

mRNA

THO: tetrameric protein complex

TREX: couple transcription & export

THO & TREX stablize mRNP formation

Recruitment of Transcription/Export Complex (TREX)

• In yeasts– Transcription

Elongation

• In mammals– During Splicing of

mRNA

Y=Yra1

S=Sub2

SR=serine/arginine rich protein (Gbp2, Hrb1)

In Yeasts

SR (Serine/Arginine-rich protein)

• In Yeasts– Gbp2– Hrb1

• In Mammals– Shuttle between the n

ucleus and cytoplasm– Regulated by phospho

rylation• Dephosphorylated form

associates with mRNA

In Mammals

Reacts with FG

RNA binding domain

Cell-Cycle Regulation

M-Phase-Promoting FactorMitotic spindle

Activation of M-Phase-Promoting Factor

References

• Journal of Biological Chemistry 280(38)32565-32568

• Journal of Biological Chemistry 279(52)53899-53902

• Essays in Biochemistry: The Ubiquitin-Proteasome System (edited by RJ Mayer &R Layfield)

• zonnc@sinica.edu.tw