Periodic table

1
Periodic table
2
Janeway 8_33
3
Hematopoietins

• Two major types of receptors M-CSFR (CSF-1),
  SCFR (C-kit) and Flt-3 internal tyrosine
  kinases.
• All others class I cytokine receptors, two
  subunits.
• IL3, IL5 and GM-CSF distinct a, common b.

4
Simplified scheme of hematopoiesis
erythrocyte
T cell
Epo
IL2, IL4
B cell
megakaryocyte
pre-T
BFU
IL2, IL4
dendritic cell
IL5, IL6
IL7
IL7 IL3
macrophage
IL3, IL6, IL11, Epo, GM-CSF
M-CSF GM-CSF
pre-B
pre-B
IL3, GM-CSF, Epo
lymphoid progenitor
neutrophil
G-CSF GM-CSF
IL3, GM-CSF, IL6
CFU-GM
CSF Flt3L
CD34
IL3, IL9, IL10
mast cell
IL3, IL4, GM-CSF
pluripotent hematopoietic stem cell
IL3, IL5, GM-CSF
myeloid progenitor CFU-GEMM
true stem cell
basophil
eosinophil
5
IL6 family

• Consists of IL6, IL11, LIF (Leukemia Inhibitory
  Factor), CNTF (Ciliary Neurotrophic Factor) and
  OSM (Oncostatin M).
• 3 subunit receptors distinct a and TWO common b
  (gp130).

6
Periodic table
7
TNF and TNFR superfamilies, 2001
Ligand
Genbank ID
Aliases
Receptor
Genbank ID
Aliases
p55-R, CD120a, TNF-R-I p55, TNF-R,
TNFRSF1A
M75866
TNF
X02910
TNFSF2, TNFA, DIF
TNFAR, p55TNFR, TNFR60, TNFR1
CD120b, p75, TNF-R, TNF-R-II, TNFR80,
LTA
X01393
TNFSF1, TNFB, LT
TNFRSF1B
M32315
TNFR2,TNF-R75, TNFBR, p75TNFR
TNFRSF3, TNFR2-RP, CD18,
LTB
L11016
TNFSF3, TNFC, p33
LTBR
L04270
TNFR-RP, TNFCR, TNF-R-III
TNFSF4
D90224
OX-40L, gp34, TXGP1
TNFRSF4
X75962
OX40, ACT35, TXGP1L
CD40LG, IMD3, HIGM1, CD40L,
TNFSF5
X67878
TNFRSF5
X60592
p50, Bp50, CD40
hCD40L, TRAP, CD154, gp39
TNFSF6
U11821
FasL, APT1LG1
TNFRSF6
M67454
Fas, CD95, APO-1, APT1



TNFRSF6B
AF104419
DcR3
TNFSF7
L08096
CD70, CD27L, CD27LG
TNFRSF7
M63928
Tp55, S152, CD27
TNFSF8
L09753
CD30LG
TNFRSF8
M83554
Ki-1, D1S166E, CD30
TNFSF9
U03398
4-1BB-L
TNFRSF9
L12964
4-1BB, CD137, ILA
TNFSF10
U37518
TRAIL, Apo-2L, TL2
TNFRSF10A
U90875
DR4, Apo2, TRAILR-1
DR5, KILLER, TRICK2A,



TNFRSF10B
AF012628
TRAIL-R2, TRICKB



TNFRSF10C
AF012536
DcR1, TRAILR3, LIT, TRID



TNFRSF10D
AF029761
DcR2, TRUNDD, TRAILR4
TNFSF11
AF013171
TRANCE, RANKL, OPGL, ODF
TNFRSF11A
AF018253
RANK



TNFRSF11B
U94332
OPG, OCIF, TR1
DR3, TRAMP, WSL-1, LARD,
TNFSF12
AF030099
TWEAK, DR3LG, APO3L
TNFRSF12
U72763
WSL-LR,DDR3, TR3, APO-3



TNFRSF12L
?
DR3L
TNFSF13
NM_003808
APRIL
TNFRSF17A
Z29574
BCMA
TNFSF13B
AF136293
BAFF, THANK, BLYS, TALL1



TNFRSF17B ?
AF023614
TACI
TNFSF14
AF036581
LIGHT, LTg, HVEM-L
TNFRSF14
U70321
HVEM, ATAR, TR2, LIGHTR, HVEA
TNFSF15
AF039390
TL1, VEGI
TNFRSF15
-
-
TNFSF16
-
-
NGFR
M14764
TNFRSF16, p75NTR
TNFSF18
AF125303
AITRL TL6 hGITRL
TNFRSF18
AF125304
AITR, GITR
TNFSF19 ?
MMAJ1386
TNFRSF19
AF173166
?
AB040434
TROY
http//www.gene.ucl.ac.uk/users/hester/tnftop.html
8
Tumor Necrosis Factor.

• TNFa, cachectin.
• Class II membrane molecule (N-terminus inside),
  in most of the cases is processed by special
  protease and secreted as 17 kD molecule.
• 230/157 aa, non-glycosylated.
• 3D structure jelly roll, protein fold
  observed in viral capsid proteins.
• Single gene, linked to class III region of the
  MHC.
• Forms homotrimers which interact with high
  affinity receptors.

9
Lymphotoxin

• Has two components, LTa and LTb and exists in at
  least 3 forms, one secreted (LTa3) and two
  membrane-bound (LTa1LTb2 and LTa2LTb1).
• LTa and LTb - closest homologs of TNF, linked to
  the same TNF/LT locus.
• LTb class II membrane molecule, targets the LTa
  subunit to membrane.
• LTa3 is similar to TNF (TNFa3) and binds to the
  same 2 TNF receptor (p55 and p75), but with lower
  affinity.
• LTa1LTb2 acts through distinct LTbR (member of
  TNF R superfamily).

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Tumor Necrosis Factor superfamily.

• Can signal activation, proliferation,
  differentiation, costimulation and cell death
  through the same receptors.
• Cytotoxic for many (but not for all) transformed
  cell lines in vitro.
• TNF, LTa, TRAIL (APO-2L) can cause necrosis of
  some (but not all) induced tumors in animal
  models.
• Involved in regulation of expression of many
  genes critical for immune response (cytokines,
  host defense genes, adhesion molecules, etc.).
• Show 25-30 similarity, aromatic residues
  responsible for trimer formation.

11
Tumor Necrosis Factor Receptor superfamily

• Extracellular portions contain several
  cystein-rich domains.
• Forms (homo) trimers which interact with ligand
  trimers through clefts.
• Intracellular portions do not possess kinase
  domains, and are not docking tyrosine kinases in
  response to binding.
• Transmit signal through distinct class of adaptor
  molecules (some of them also trimeric).
• Can transmit two types of signals activation
  (proliferation, differentiation, costimulation)
  and programmed cell death (PCD).

12
TNF Receptor/Ligand structures

• TNF, TRAIL (APO-2L) jelly-roll structure.
  TRAIL3 is stabilized by Zn ion.
• Extracellular portions of receptors contain
  several cystein-rich domains.
• Two complexes solved (extracellular domains of
  TNFRp55 bound to LTa) and TRAIL bound to DR5.
• In both cases homotrimers interact with ligand
  trimers through clefts (groves).
• Intracellular portions do not possess kinase
  domains, and are not docking tyrosine kinases in
  response to binding.
• Transmit signal through distinct class of adaptor
  molecules (some of them also trimeric).

13
Apoptosis (programmed cell death).

• Normal physiological process (embryonic
  development, cell renewal).
• Associated with irreversible disruption of
  nuclear structures, chromatin and DNA changes in
  cytoplasmic organelles and cell membrane. As a
  result, fragments of dying cells are recognized
  and digested by phagocytes.
• Necrosis is another form of cell death, is not
  associated with the orderly collapse of cellular
  structures and necrotic cells are not digested.

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Two apoptotic pathways

• Instructive apoptosis external signal,
  transmitted through death receptors.
• Intrinsic (damage induced) pathway. Mediated by
  mitochondria.
• Withdrawal of growth factor or cytokine signaling
  is sensored by mitochondria and may result in
  apoptosis by intrinsic pathway.

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Types of apoptosis
16
Apoptosis/Evolution/Genes

• Model organism nematode (C. elegans). Each gene
  (19, 099) and each of 959 somatic cells are
  known.
• 3 genes involved in regulation of apoptosis
  CED-3, CED-4 pro-apoptotic CED-9
  anti-apoptotic.
• CED-3 is a founding member of caspase family.

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Death receptors

• System to control/activate extrinsic pathway of
  programmed cell death (instructive apoptosis)
• Responds to extracellular protein factors
  cytokines.
• Involves cellular receptors belonging to TNF R
  superfamily.
• Two most studied death receptors TNFRp55 and
  Fas (APO-1, CD95).
• Death receptors contain in their intracellular
  part a short domain which is necessary and
  sufficient to signal apoptosis.

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Caspases

• Proteases with Cysteine in the active center,
  cleave after Asp.
• Synthesized as inactive precursors.
• Activated due to recruitment into multiprotein
  complexes.
• Recruitment depends on homotypic protein-protein
  association with molecules containing caspase
  activation and recruitment domain (CARD).
• At least 12 caspases in humans.
• Regulatory (proximal) caspases (8 and 9)
• Effector (executioner) caspases (3 and 7).

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Some caspases
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Caspase targets

• Multiple targets.
• Lamin A essential component of nuclear
  structure.
• PARP (poly(ADP-ribose) polymerase, component of
  DNA repair system.
• U1-70kd splicing factor.
• DNA-PK component of DNA repair and Toll
  signaling.
• Rb tumor suppressor, cell cycle regulator.
• H1 histone essential for higher order chromatin
  organization.
• CAD caspase-activated DNAse, at least partly
  responsible for DNA cleavage in apoptotic cells.

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Initiation mechanisms (both pathways)

• Caspase-dependent
• Proenzyme recruitment, oligomerization,
  proximity-induced catalytic activation.

22
Caspases in instructive and intrinsic apoptotic
pathways.

• Caspase 8 the central caspase of the
  instructive pathway
• Caspase 9 the best studied caspase of intrinsic
  pathway.
• Caspase 3 belongs to both pathways.
• C. elegans gene Ced4 is a homolog of mammalian
  caspase 3.
• The two pathways are interconnected caspase 8
  cleaves BID and affects the intrinsic pathway.
• Knock-out of caspase 8 in mice results in early
  embryonic lethality.

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TNF receptor/ligand 3D structutre


View A

A

A

TNFRp55

LTa
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Death receptors/ligands

• TNFRp55, Fas, DR3, DR4, DR5, DR6
• TNF, LTa, Fas-L, TWEAK, TRAIL (APO-2L)

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Death receptors, subfamily of TNF receptor
superfamily.

• Signaling depends on receptor aggregation (may
  occur in the absence of ligands).
• To prevent ligand-independent signaling, some
  receptors use special class of molecules,
  silencers of death domains (SODD), which
  dissociate upon ligand binding.
• Within seconds after receptor engagement the
  death activating signaling complex (DISC) is
  formed on the intracellular portions of the
  receptors.
• Death signal is transmitted through recruitment
  of FADD, and pro-caspase 8 (Fas, DR4-5) or TRADD,
  FADD, pro-caspase 8 (TNFRp55).
• Activation signal is transmitted through RIP,
  MAPK3, IKK, NFkB or TRAF2, JNK and AP1.

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Procaspase-8 processing at the DISC
CD95L
CD95
caspase-8 prodomain
C
C
x
C
p12-c-FLIPL
p10
x
C
p18
active caspase-8
procaspase-8
c-FLIPL
FADD
c-FLIPS
27
Non-receptor proteins containing DD
DD
TRADD
DD
DED
FADD
DD
Ser-Thr kinase domain
RIP
DD
CARD
RAIDD
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Fas (Apo-1, CD95)

• Initially erroneously described as a component of
  TNF R system.
• Type I transmembrane protein (N-terminus
  outside), 48 kD, 319 aa.
• Contains death domain (ca 80 aa).
• Recruits FADD which in turn recruits pro-caspase
  8.
• Does not activate genes.
• Expressed on activated T and B cells and involved
  in downregulation of immune responses.
• KO mice develop splenomegaly, lymphoproliferative
  and autoimmune disorders. Lpr mice is a leaky Fas
  KO.

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TRAFs.

• Associated with TNFR superfamily.
• Initially described for TNFRp75.
• Non-DD receptors use TRAFS (direct binding)
• Some of DD-containing receptors also use TRAFs
  (without direct binding).

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TNFRp55 signaling

• Can signal both apoptosis and gene activation
• Recruits TRADD, does not directly recruit FADD.
• TRADD recruits FADD and promotes recruitment and
  autoactivation of procaspase 8.
• TRADD recruits RIP and TRAFs.
• RIP initiates kinase cascade to activate NFkB
• Alternatively, RIP recruits RAIDD, which in turn
  recruits procaspase 2.
• TRAF2 initiates MEK/JNK pathway to activate AP1

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TNFRp55 signaling
TNFa3 or LTa3
TNF-RI (p55)
RIP
FADD
Death domain
Death domain
Death domain
Death domain
pro-caspase 8
CARD
TRADD
TRADD
kinase
TRAF 2
effector domain
MAP3K
caspase 8
JNK
c-Jun
caspase cascade
NF-kB activation
AP-1
Gene Activation
Apoptosis
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TRAIL/APO-2L

• DR3-5
• DR3 signals as TNFRp55 (RIP, TRAF)
• DR4-5 as FAS (FADD, casp 8), no RIP
• FADD universal death adaptor

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Protection from apoptosis

• Same DD receptors can launch protective signal
• Breaks at multiple points in the pathway (FLIP,
  IAP).
• Decoy receptors (DcR1-2 for TRAIL DcR3 for FasL
  and LIGHT viral decoys).
• Decoys with partial function (DcR2 can induce
  NFkB).

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Periodic table
35
Chemokines (chemotactic cytokines)

• Four subfamilies, overall more than 100 members.
• Classification based on two first cysteins
• Two large families a or CXC and b or CC.
• Two additional types g or C and d or CX3C.
• Receptors 7 TMD, G-protein coupled.
• Two major receptor families CXCR1-N and CCR1-N
• Most of chemokines interact with multiple
  receptors, and most of receptors respond to
  multiple chemokines.

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Chemokine specificity
CXCR1 --- IL8 gtgt GCP2 gtgt NAP2, ENA78
neutrophil
CXCR2 --- GRO, NAP2, IL8, ENA78, GCP2
resting T cell
CXCR4 --- SDF1
CXCR3 --- IP10, MIG, I-TAC
CXCR5 --- BCA-1
activated T cell
CX3CR1 --- Fractalkine
CCR1 --- MIP1a, RANTES, MCP2-4
B cell
CCR2 --- MCP1-5
CCR3 --- Eotaxin1-2, RANTES, MCP2-4, MIP1a
monocyte
CCR4 --- TAPC, MDC
CCR5 --- MIP1a, MIP1b, RANTES
basophil
CCR6 --- LARC
CCR7 --- ELC, SLC
eosinophil
CCR8 --- I-309, TARC, MIP1b
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Cytokines and cancer
Role of chemokines in metastasis

• Chemokine receptors CXCR4 and CCR7 are highly
  expressed in human breast cancer cells
• Ligands for these receptors CXCL12 (SDF-1a) and
  CCL21 (6Ckine) are abundantly expressed in the
  primary sites of breast cancer metastasis

Picture from L.L.Liotta, Nature 410 24-25 2001.
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Clinical use of cytokines

• Interferons a (Roferon, Alferon-N, Intron
  A) antiviral therapy (chronic Hepatatis B and
  C), hairy cell leukemia.
• Interferon b (Betaseron) multiple sclerosis.
• G-CSF (Neupogen) supportive treatment for bone
  marrow transplantation.
• Interferon g (Actimunne) chronic
  granulomatosis.
• Epo (Procrite) kidney disorders.
• GM-CSF, IFN-g, IL2, TNF all toxic when applied
  systemically.

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Cytokines in cancer therapy.

• Interferons a (Roferon, Alferon-N, Intron
  A) hairy cell leukemia, CML.
• G-CSF (Neupogen) supportive treatment during
  cancer chemotherapy.
• Epo (Procrite) supporting treatment during
  cancer chemotherapy.
• IL2, GM-CSF and others for maintaining primary
  cell cultures used for adoptive immune therapy.
• IL2 (Proleukin) in cancer therapy (metastatic
  renal cancer).
• TNFIFNg chemotherapy effective when applied
  locally (regional perfusion).

40
Promising clinical trials for cytokines.

• TRAIL (APO2-L) in cancer therapy.

41
How specificity is brought about?

• In spite of understanding some molecular details
  in cytokine signalling, the specificity in the
  activation process is NOT fully understood.

42
About specificity

• Many promoters integrate complex multiple signals
• Everything must be in place at the right time
  and in correct orientation

MEKK -gt ERK
JNK
Elk
AP1
MAP3K

TATA
43
Acknowledgements

• D. Kuprash (Moscow)
• A.Shakhov (Frederick)
• R. Kirken (Houston)
• I. Lavrik (Heidelberg)
• R.Kazaryan (Moscow)