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
expression
8 X symmetry

90 nm
 FG Repeat/non-
structure

Nucleoporins
FG Repeat
Binding Site

FG Repeat
Binding Site
 Nucleoporins: glycosylated &
hydrophobic
• Mammals • Yeasts
– FXFG – GLFG

Most of them located symmetrically at NPC,

whereas only a few are located asymmetrically.
Blocked with the
hydrophobic
compound
hexane diol
 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
Importin
Exportin

HECT: E3 ubiquitin ligase domain

 Sequential Events

Multiple Sites for Receptor Recognition

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

Exportin/cargo/GTP-Ran Importin/substrate in cytosol

RanGAP &RanBP1&RanBP2 In the presence of GDP-Ran
Exportin<<<<<>>>>>Cargo Importin<<<<>>>>Substrate
Interconnectivity of Transcription
and the Ubiquitin-Proteasome
System
Dynamic: Exchange of
Complexes
Regulation of levels of transcription
factors
∀ β-catenin
• c-myc
• Nuclear Hormone Receptors
 Nuclear Hormone Receptors
•Steroid Hormone • Non-Steroid Hormone
Receptors Receptors
–In cytoplasm with – In nucleus at hormone
chaperone response elements as
•Estrogen a heterodimer with
•Androgen RXR
•Progesterone • Vitamin D
•Glucocorticoid • Retinoic Acid
• Peroxisome
Proliferator-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
Chromosomal Architecture
• Methylation
• Ubiquitin-Proteasome Pathway
 Chromatin-Modifying Enzymes
• ATP-dependent • Histone-modifying
nucleosome enzymes
remodelling enzymes – Histone
– SW1/SNF acetyletransferase
(HAT)
 Coactivators for Nuclear Hormone
Receptors
• P160
• Histone Aceyltransferase (HAT)
• Histone Arginine Methyltransferases
• Nucleosome Remodeling Complex
• Multisubunit Mediator Complex: bridge
nuclear hormone receptors and the basal
transcriptional machinery
Special Ubiquitinylation

Not for degradations but for

molecular interactions
Mono-
Ubiquitin
trans

H3
Phosphorylation Me Ac
H3
Ubiquitin as a sorting Signal

GH receptor Endocytosis
EGF receptor internalization
Glutamate receptor
etc
Proteasome In Eukaryotes
Threonine Proteases
Beta1, beta2, beta5 with catalytic activity.
Beta1: post-glytamyl-peptide-hydrolyzing; beta2:
trypsin like; beta5:chymotrypsin like.
 catalytic
Beta 1, 2 & 5
Regulatory Factors for Proteasome
Assembly
• Positive • Negative
– Hsp90 – PAAF-1 (proteasomal
ATPase-associated
factor-1)
Closed Open
Activator & Inhibitor

19S=PA700
PA28
PI31
PI31
Phosphorylation
 19S
• Base • Lid: Non-ATPase
– Rpt1-Rpt6: ATPase – 8 subunits
– Non-ATPase – Rpn10: a hinge between
• Rpn1 the base and lid
• Rpn2 – Rpn11: An intrinsic Zn2+-
dependent
metallopeptidase to release
ubiquitin and peptides for
20S proteasome
– Rpn4: Binds and acts as a
transcriptional activator
 PA28 (Proteasome Activators)
• Alpha/beta • Gamma
– In immune tissues – For trypsin-like activity
– cytoplasmic • In brain
• In nucleus
 Immuno-Proteasomes
• Page 43
Fool the substrates for E3

RING
Virus

HECT
 E3
RING
• RING (Really Interesting New Gene)
– Two zinc atoms
– No directly associated with ubiquitin

– Such as Mdm2 to ubiquitinize β-arrestin

– C-Cbl
 E3
HECT

Homologous to E6-AP C-terminal

Recognize proline rich motifs
350 a.a. residues
Ubiquitin Pathway
 Ubiquitin: 76 a.a. polypeptide
K
29

K
48

K
63
Ubiquitin
 E3
• SCF (Skip-Cullin-F- • Anaphase-Promoting
box) Complex (APC;
– Phosphorylation of he cyclosome) contains
target protein often a RING-finger protein
triggers ubiquitylation (Apc11), an E2 and a
cullin (Cdc20/Hct1).
SCF
RING: Roc1

ECS 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
 Lipid

Protein

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
ψKXE
Ψ:hydrophobic
 Substrates for Sumoylation
• RanGAP ψKXE/D
• PCNA TKET
• Smad4 VKYC
• VKDE

Proteases for SUMO

• SENP
• SuPR1 & Axam2 are splice variants of
Senp2
 unique

=ubiquitin like protease 1=cysteine

protease family
 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
• IκB
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 • In mammals
– Transcription – During Splicing of
Elongation mRNA
Y=Yra1
S=Sub2
SR=serine/arginine rich
In Yeasts
protein (Gbp2, Hrb1)
 SR (Serine/Arginine-rich
protein)
• In Yeasts • In Mammals
– Gbp2 – Shuttle between the
– Hrb1 nucleus and cytoplasm
– Regulated by
phosphorylation
• Dephosphorylated form
associates with mRNA
In Mammals
 Reacts with FG

RNA binding
domain
Cell-Cycle Regulation

M-Phase-Promoting Factor
Mitotic 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