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Phospholipids, Phosphoinositols & Eicosanoids. Phospholipids, Phosphoinositols & Eicosanoids. Common types of Phospholipids:. Phospholipids, Phosphoinositols & Eicosanoids. Second messenger released through hydrolysis by phospholipases and/or

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Phospholipids phosphoinositols eicosanoids1
Phospholipids, Phosphoinositols & Eicosanoids

Common types ofPhospholipids:


Phospholipids phosphoinositols eicosanoids2
Phospholipids, Phosphoinositols & Eicosanoids

Second messenger

  • released through hydrolysis by phospholipases and/or

  • generated through the actions of lipid kinases

Phospholipases:

Phosphatidylinositol-kinases:


Phospholipids phosphoinositols eicosanoids3
Phospholipids, Phosphoinositols & Eicosanoids

Phospholipases:

  • PLA2:

    • Cytoplasmic form (90 kDa) is regulated through nM Ca++ (Annexins) and phosphorylation; AA specific => signaling function

    • Secreted form (pancreas, 14 kDa) is also Ca++ dependent (mM range)=> digestive function

  • PLC: coupled to a variety of (growth factor) receptors:

    • PLC is activated through GPCRs (Gq) => binding enhances its catalytic activity and in return the GTPase activity of Gq (similar to GAP function in ras signaling)

    • PLC couples with its SH2 domains directly to growth factor receptors (EGFR, PDGFR) or the TCR, where it is activated through tyrosine phosphorylation


Phospholipids phosphoinositols eicosanoids4
Phospholipids, Phosphoinositols & Eicosanoids

Both phospholipases yield finally arachidonic acid (see below), in addition, PLC activity also produces DAG and IP3:

  • DAG: remains membrane bound; diacylglycerol kinase phosphorylates DAG to generate phosphatidic acid which functions as a substrate for PLA2.

    Phosphatidyl-serine (PS), Ca++ and DAG activate PKC on the plasma membrane

  • IP3: see Ca++ signaling!!

    • Glucocorticoids: inhibit PLA2 by transcriptionally inducing Lipocortin, a protein which binds to PLA2 and blocks its activity.

    • Phorbol esters: strongest known tumor promotors; mimic DAG => bind PKC and activate it. Also potent activator of Ca2+ influx, MAPK pathway etc.


Phospholipids phosphoinositols eicosanoids5
Phospholipids, Phosphoinositols & Eicosanoids

Lipid kinases:

  • PI3-kinase:

    • binds to and becomes tyrosine phosphorylated in response to activation of growth factor receptors or immune receptors

    • 85 kDa regulatory subunit (pY) and a 110 kDa catalytic subunit

    • regulatory subunit contains SH2 and SH3 domains

    • PIP3-phosphates can bind to the pleckstrin homology (PH) domain of Akt

      => Akt activation => phosphorylation of BAD, which dissociates from the antiapoptotic protein bcl-2 => inhibition of apoptosis

  • Wortmannin: fungal metabolite, potent, irreversible inhibitor of PI3Kinase

  • Ly290004: synthetic compound, blocks ATP binding site of PI3Kinase


Arachidonic acid metabolism
Arachidonic Acid Metabolism

  • Eicosanoids:

    collective name for derivatives of arachidonic acid (=5,8,11,14 - eicosatetraenic acid)

    • AA is mainly generated through the action of PLA2 and DAG-lipase.

    • Rapidly metabolized by cyclooxygenase and lipoxygenase into

      prostaglandins and leukotrienes:


Arachidonic acid metabolism1
Arachidonic Acid Metabolism

  • Prostaglandins:

    • First observed in seminal fluid => name

    • Structure of cyclopentane ring defines letter

    • Double bonds in side chains account for number

    • Greek letter refers to the spatial position of the OH-group at C-9

Initial step in PG synthesis catalyzed by PGH-synthase which has dual enzymatic activity:

cyclooxygenase (closes ring =>PGG2)

and

peroxidase (=> 15-OH)


Arachidonic acid metabolism2
Arachidonic Acid Metabolism

Biological functions of PGs:

  • Vascular toneRelaxation: PGs E1, E2, F2 and I2

    Constriction: PGs F2, TxA2

  • Platelet aggregationIncrease: PGs E1, TxA2

    Decrease: PGs E2, I2

  • Uterus toneIncrease: PGs E1, E2, F1

  • Bronchial muscleConstriction: PGFs

    Relaxation: PGEs

  • Gastric secretionInhibition: PGs E1, E2, I2

  • Temperature and painIncrease: PGEs


Arachidonic acid metabolism3
Arachidonic Acid Metabolism

  • Leukotrienes:

    • First found in leucocytes; contain 3 conjugated double bonds

    • Lipoxygenase generates Hydroperoxyeicosatetraenoic acid (HPETE)

    • LTC4, D4 and E4 mediate allergic reaction: Slow Reacting Substance of Anaphylaxis (SRS-A) => mediates anaphylactic shock 10,000 fold more potent than histamine!!! => constricts bronchi, dilate blood vessels

    • LTB4 is a very strong chemoattractant for macrophages


Growth factor receptors
Growth Factor Receptors

  • Many growth factors (EGF, PDGF, IGF-1, CSF-1, ...) signal through receptors withintrinsic tyrosine kinase activity

  • Common features:

    • Large, glycosylated ligand binding domain

    • Single hydrophobic transmembrane domain

    • Activation occurs through ligand mediated oligomerization

    • Undergo ligand induced downregulation by internalization

    • Cytoplasmic tyrosine kinase domain:

      • most highly conserved region

      • GlyXGlyXXGlyX(15-20)Lys

        Lys is critical for ATP binding - mutation renders receptor kinase inactive, which abrogates all cellular responses => signaling depends on tyrosine phosphorylation

        of receptor and cytoplasmic substrates

      • Tyrosine kinase receptors also bind and activate cytoplasmic tyrosine kinases

    • Autophosphorylation sites:

      • conserved in the C-term of each receptor class

      • autophosphorylation does not effect Km of receptor kinase activity

      • provide docking sites for SH2 domain containg signaling proteins


Growth factor receptors1
Growth Factor Receptors

  • Three subclasses:

    • Class I: two Cys-rich region in the EC, monomeric ligand

      EGF-R, erbB2, erbB3, erbB4 (heregulin receptors)

    • Class II: heterotetrameric: 2  and 2  chains stabilized through S-S bonds: monomeric ligand

      Insulin-R, IGF-1-R

    • Class III: Repeats of mmunoglobulin-like structure, dimeric ligand

      FGF-R, NGF-R, PDGF-R, CSF-1-R, c-kit


Growth factor receptors2
Growth Factor Receptors

Signaling through Adapter proteins:

  • grb2: adapter with one SH2 domain which binds PY residue on RTK, and two SH3 domains which bind to

  • Sos: “Son of Sevenless” (mutation in drosophila prevents development of the R7 photoreceptor cell). Functions as a GEF to facilitate GTP loading of

  • ras: GTP binding protein, farnesylated; protooncogene, provides a docking site on the plasma membrane for raf.

  • ras-GAP: negative regulator of groth factor signaling: promotes GTP hydrolysis through the GTPase activity of ras.


Growth factor receptors3
Growth Factor Receptors

Signaling through Adapter proteins:

  • raf: ser/thr kinase of the MAPKKK/MEKK family (MEKK does normally NOT phosphorylate MEK, but rather MKK4/7 => Stress pathway); requires context of plasma membrane for activation (mixing GTP-ras and raf in a test tube fails to activate raf) => raf likely to be phosphorylated at the plasma membrane. Activated raf phosphorylates...

  • MEK: Dual specificity kinase (in case of Stress pathway: SEK) phosphorylates ERKs or MAPKs on tyr and thr ->

  • ERKs: migrate to the nucleus where they phosphorylate transcription factors such as fos and jun; also feedback loop to other signaling molecules



Cytokine receptors
CytokineReceptors

  • “Classical” hormones:

    • produced by cells organized into endocrine organs,

    • often referred to as “endocrine signal molecules”

    • target cells usually distant from the site of synthesis

    • hormone carried by blood stream from producing gland to target cells

    • signal through receptors coupled to G-proteins (e.g. epinephrine receptor), ion-channels

    • (e.g. acetylcholine receptor) or receptors with intrinsic enzymatic activity

  • Cytokines:

    • single producing and effector cell

    • only affect target cells in close proximity (autocrine or paracrine)

    • almost exclusively involved in regulating immunological processes

    • sometimes subdivided into different groups based on their origin (lymphokines, monokines, interleukines)

    • often carry several (old) names based on their multiple biological functions: e.g. Lymphocyte Activating Factor (LAF) = Mitogenic Protein (MP) = T Cell Replacing Factor III (TRF-III) = B Cell Activating Factor (BAF) = B cell Differentiation Factor (BDF) = INTERLEUKIN 1


Cytokine receptors1
CytokineReceptors

  • Basic characteristics:

    • only one copy of encoding gene per haploid cell (~20 different IFN’s, but each encoded by a distinct gene)

    • genes segmented into 4 or 5 exons (exceptions are IFN and IFN: no introns)

    • mature protein usually between 8 and 25 kDa

    • barely any structural resemblances

    • often N-glycosylated

    • often form oligomers

    • some carry signal sequence in precursor

    • expression is tightly regulated on a transcriptional level

    • generally pleiotropic

    • usually highly species specific (up and down)

  • Multiple (old) classifications:

    • Based on origin (Lymphokines, Monokines..)

    • Based on action (inhibitory, stimulating, antiviral, chemotactic...)

    • Based on composition of receptor (single-chain vs. multi-chain)


Cytokine receptors2
CytokineReceptors

  • Current nomenclature based on structure of receptors:

    • Type I cytokine receptors = hematopoietin receptor family:receptors contain W-S-X-W-S motif in the C-terminus

      • IL-2 R, IL-3 R, IL-4 R, IL-5 R, IL-6 R (has also Ig-like domain), IL-7 R, IL-9 R, IL-11 R

        IL-13 R, IL-15 R, GM-CSF R, EPO R, G-CSF R (has also Ig-like domain)

    • Type II cytokine receptors = Interferon receptor family:receptors contain IRH1 (200aa extracellular) and IRH2 (50 aa cytoplasmic) domain

      • IFN R, IFN R

    • Type III cytokine receptors = TNF receptor family: receptors contain 4 Cys rich regions in extracellular domain

      • TNF R, TNF=LT R, NGF R (trk), fas, CD40

    • Type IV cytokine receptors = Immunoglobulin family: receptors contain an Ig like repeat in the extracellular domain

      • IL-1 R, M-CSF R (c-fms), SCF R = steel factor R (kit) (tyrosine kinase activity), (IL-6 R) (has also W-S-X-W-S motif), (G-CSF R)

    • (Chemokine receptors):

      • C-X-C subgroup (-family): IL-8, PF4, TG

      • C-C subgroup (-family): RANTES, MCAF, MIP-1


Cytokine receptors3
CytokineReceptors

Signal Transduction:

Receptors lack intrinsic catalytic activity but associate w/ cytosolic enzymes

STAT: Signal transducer and activator of transcription

  • contain SH2 domains

  • become tyrosine phosphorylated after stimulation

  • 6 family members

  • homo or heterodimerize

  • translocate to nucleus and bind enhancers

    JAK: Janus kinase

  • large cytoplasmic tyrosine kinases (130-140 kDa)

  • NO SH2 or SH3 domains

  • kinase-like domain


Ser thr kinase receptors
Ser/Thr-kinaseReceptors

Transforming Growth Factors = TGFs:

  • Murine sarcoma virus infected cells can not bind EGF => cells produce a growth factor that competes for EGF binding (=sarcoma growth factor, SGF).

  • SGF promoted anchorage independent growth (reversible) => first evidence that transformed cells produce their own growth factor!

  • SGF was found to consist of two subunits:

    TGF: EGF competitor, binds and signals through the EGF receptor, potent mitogen, but does not support anchorage-independent growth

    overexpressed in epithelium of psoriasis patients (=> hyperproliferation of keratinocytes)

  • TGF: acts through distinct receptor, it also is a potent mitogen, but it also does not support anchorage-independent growth (only combination does!)

    TGF- receptors I and II:

  • external ligand binding domain and cytosolic serine/threonine kinase activity.

  • betaglycan: proteoglycan required for TGF binding


Ser thr kinase receptors1
Ser/Thr-kinaseReceptors

Signal Transduction:

SMADs:

  • 8 members - conserved MH1 and MH2 domain

  • rapidly phosphorylated in response to TGF (2,3) or BMP (1,5,8)

  • Smads function in heteromeric complexes

  • “common” Smad = Smad-4 (Smad-4 required for all Smad signaling)

  • translocate to the nucleus

  • phosphorylated by the receptor itself

  • phosphorylation motif: SSXS

  • Smad1 potentially also activated by MAPK

  • Mutations of Smads and /or TGFR are found in 90% of colon cancers


Nf k b
NFkB

  • originally identified as a transcription factor binding an enhancer (B)in the -light chain immunoglobulin gene

  • Activated by a variety of (proinflammatory) signals (IL-1, TNF, Phorbol esters...)

  • Homo-or heterodimer composed of p50 and/or p65 subunits

  • retained in its inactive form in the cytoplasm by the inhibitory protein IB

  • dissociation of NFB from IB activates NFB’s DNA binding capabilities

  • NFB/ IB association is regulated by serine phosphorylation of IB!

  • Phosphorylated IB does not dissociate from NFB, rather is marked for degradation (NFB activation can be inhibited by protease inhibitors!)

  • Phosphorylation of IB through IB-kinase (complex >600 kDa)

  • IKK: two kinase subunits: IKK and IKK homo- or heterodimers IKK (NEMO): no kinase activity, required for complex assembly = regulatory subunit


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