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Structures in the B7/CD28 family. Structures are modeled on the crystal determinations. Loops have been added to one end of the IgV domains to emphasize the orientation of the CDR-like loops and their interaction with ligand or lack thereof.
EPSPs: excitatory post-synaptic potentials
IPSPs: inhibitory post-synaptic potentials
What kinds of hormone are there?What kinds of hormone are there?
Known Hormonal Classes
• Proteins & peptides
• Lipids (steroids, eicosanoids)
• Amino acid derived (thyronines, neurotransmitters)
• Gases (NO, CO)
chemcases.com/olestra/ images/insulin.jpg
chem.pdx.edu/~wamserc/ChemWorkshops/ gifs/W25_1.gif
website.lineone.net/~dave.cushman/ epinephrine.gif
What is a hormone receptor?What is a hormone receptor?
Hormone Receptors are cellular proteins that bind with high affinity to hormones & are altered in shape & function by binding; they exist in li
mited numbers.
Binding to hormone is noncovalent & reversible.
Hormone binding will alter binding to other cellular proteins & may activate any receptor prote
in enzyme actions.
What are the main types of receptors?What are the main types of receptors?
Membrane ReceptorsImbedded in target cell membrane; integral proteins/glycoproteins; penetrate through membrane
For protein & charged hormones (peptides or neurotransmitters)
3 major groups: Serpentine = 7 transmembrane domains, Growth factor/cytokine = 1 transmembrane domain, Ion channels
What are the main types of receptors?What are the main types of receptors?
Nuclear ReceptorsNuclear proteins that usually act in pairs & bind to specific Hormone Recognition Elements (HREs) = sequences on the DNA in the promoter regions of target genes
For small, hydrophobic molecules (steroids, thyroid hormones)
G – G protein, GTP – guanosine triphosphate, PLC - phospholipase C, PLD - phospholipase D, PLA2 - phospholipase A2, AC – adenylyl cyclase, IP3 – inositoltriphosphate, DG – diacylglycerol, cAMP – cyclic adenosine monophosphate, FFA – free fatty acids, PKC – protein kinase C, PKA – protein kinase A, PKCaM – Ca2+ and calmodulin dependent protein kin
ase, PP – phosphoprotein phosphatase
Gs/i/o/x – G proteins, CaM – calmodulin, GTP – guanosine triphosphate, ATP –
adenosine triphosphate, ADP – adenosine diphosphate, cAMP – cyclic adenosine monophosphate, 5´-AMP - 5´-adenosine monophosphate, PKA – protein kinase A, PKC – protein kinase C
Signal transduction: Adenyl cyclase system
Signal transduction: Phosphoinositide system
PI-PLC - phospholipase C specific for phosphoinositides, PIP2 - phosphatidylinositol-4,5-biphosphate, IP3 ‑ inositol-1,4,5-triphosphate; DG - diacylglycerol; PKC – protein kinase C
AR – adrenoceptor, G – G protein, PI-PLC – phosphoinositide specific phospholipase C, IP3 – inositoltriphosphate, DG – diacylglycerol, CaM – calmoduli
n, AC – adenylyl cyclase, PKC – protein kinase C
Gs/i/o/x – G proteins, CaM – calmodulin, GTP – guanosine triphosphate, ATP – adenosine triphosphate, ADP – adenosine diphosphate, cAMP – cyclic adenosine monophosphate, 5´-AMP - 5´-adenosine monophosphate, PKA – protein kinase A, PKC – protein kinase C
GC – guanylyl cyclase, CaM – calmodulin, NO – nitric oxide, nNOS – nitric oxide synthase, ATP – adenosine triphosphate, cGMP – cyclic guanosine monophosphate
Cell survival
Cytoskeletal rearrangements
PI-3 kinase
(Yao and Cooper, 1995)
Phosphatidylinositol–3 kinase (PI-3 kinase)
• Models of estrogen action. In the “classical” pathway of estrogen action (i), estrogen or other selective estrogen receptor modulators (SERMs) bind to the estrogen receptor (ER), a ligand-activated transcription factor that regulates transcription of target genes in the nucleus by binding to estrogen response element (ERE) regulatory sequences in target genes and recruiting coregulatory proteins (CoRegs) such as coactivators. Rapid or “nongenomic” effects of estrogen may also occur through the ER located in or adjacent to the plasma membrane (ii), which may require the presence of “adaptor” proteins, which target the ER to the membrane. Activation of the membrane ER leads to a rapid change in cellular signaling molecules and stimulation of kinase activity, which in turn may affect transcription. Lastly, other non-ER membrane-associated estrogen-binding proteins (EBPs) may also trigger an intracellular response (iii).
• Risk factors can be distinguished in terms of their ability to cause breast cancer directly, through genetic damage, or by altering hormonal metabolism. Vulnerability factors prolong the duration of breast cell growth, while contributing factors can distort hormone levels.
• Bifunctional pathways to breast cancer. Abbreviations: E2, 17ß-estradiol; E1, estrone; OHE1, hydroxyestrone; ER, estrogen receptor. In the bifunctional pathway, the E2 metabolites affect cell proliferation and breast cancer development either directly via receptor-independent mechanisms involving structural/functional alterations in DNA, or indirectly via receptor-dependent mechanisms involving phenotypic growth regulation. Both mechanisms eventually upregulate aberrant proliferation and development of breast cancer (37).
Figure 19-34 Fluctuating Levels of Mitotic Cyclin and MPF During the Cell Cycle
Figure 19-38 Role of the Rb Protein in Cell Cycle Control
Figure 19-39 Role of the p53 Protein in Responding to DNA Damage
Apoptosis
• Apoptosis (1972)– Greek word “falling off”
• Built-in (programmed)mechanism)
• or self-destruction-suicide
• Type of programmedcell death based uponmorphological features
Programmed cell death during development. Programmed cell death is involved in forming structures such as the digits of the hand (a), deleting structures such as nearly all of an insect's larval components (b), controlling cell numbers in, for example, the nervous system
(c) and eliminating abnormal cells such as those that harbour mutations (d).
Jacobson et al (1997) Cell, Vol. 88, 347–354,
Apoptosis plays in an important role in normal developmental processes
Studies on the development of the nervous system showed that in the process of assembling sensory fields, neurons are eliminated by orderly cell death in order to tailor sensory input to environmental stimuli (elimination or transplantation of limbs as key examples).
A cancer cell (mauve) undergoing apoptosis
Comparison of cell death by necrosis and apoptosis
Apoptosis
Cell directed "suicide" Volume loss Membrane blebbing Chromatin condensation Cytochrome CCaspaseDNA fragmentation Apoptotic bodies Non inflammation
Known Apoptotic Stimuli
• Withdrawal of NGF
• Etoposide
• Actinomycin D
• UV radiation
• Staurosporin
• Enforced m-Myc expression
• Glucocorticoids
Physiological Relevance of Apoptosis
• Embryonic Development
• Regulation of/by Immune System
• Negative Selection
• CTL Killing (eg. Immune surveillance, viral infections)
• Terminating Active Immune Response
• Tight Regulation of Cell Number (eg. BM, GI, Uterus, Skin)
• Compensatory Response to Cell Stress
• Intrinsic Pathway (e.g. GF removal, XRT, Chemo.)
• Extrinsic Pathway (e.g. FAS.Ag, TNF-R Activation)
• Senescence (ageing)
Evidence Needed to Verify the Occurrence of Apoptosis
• Morphological changes – Formation of apoptotic bodies and blebbing
• DNA fragmentation– Genomic DNA: Ladder appearance – Positive TUNEL*
• Effectiveness of endonuclease inhibitors and protein synthesis inhibition
• Annexin V positive labeling• TUNEL- Terminal UDP Nick-End labeling
Morphology of Apoptosis– cell shrinkage– extracellular exposure of phosphatidylserine– shows boiling and blebbing – chromatin condenses*-most characteristic feature– DNA is degraded into oligonucleosomal fragments– disassembly into apoptotic bodies
• membrane bound, contains portions of the nucleus and various organelles
– phagocytosis by neighboring cells
Does not elicit inflammation-hallmark of apoptosis
mitochondria
Caspase-3 activation via tumor necrosis factor (TNF) family receptors (for example, Fas), FADD (Fas-activated death domain protein) and caspase-8 represents the extrinsic pathway (blue), whereas caspase-3 activation via the mitochondrial release of cytochrome c and Apaf-1–mediated processing of caspase-9 represents the intrinsic pathway (red)3.
For clarity, not all of the players are shown. Procaspase-3 is shown as a PAC-1–sensitive dormant single-chain precursor with an N-terminal prodomain (Pro). During apoptosis, caspase-3 assembles as an active p17-p12 heterotetramer after proteolytic processing between the p17 and p12 subunits (at Asp175) and removal of the prodomain2.
PAC-1 is proposed to regulate the Asp-Asp-Asp (DDD) safety catch at amino acids 179–181 in procaspase-3, consequently inducing a conformational change that leads to proteolytic processing into the active p17 and p12 subunits1.
Cys163 is the catalytic cysteine in the active site of caspase-3; the sequence shown illustrates its proximity to the DDD safety catch and DDM motif. Although caspase-7 (not shown) is believed to be a downstream caspase, its position relative to caspase-3 in apoptosis pathways is unclear.
Functional homologues of caspases and caspase regulators across species are indicated by the same colour. Caspase-9 in mammals and Dronc in the fruitfly Drosophila melanogaster are initiator caspases, whereas caspase-3 and -7 in mammals and Drice in fruitflies belong to the class of effector caspases.
CED-3 (cell-death abnormality-3) in the nematode worm Caenorhabditis elegans functions both as an initiator and effector caspase.
The inhibitor of apoptosis (IAP) proteins suppress apoptosis by negatively regulating the caspases, whereas SMAC (second mitochondria-derived activator of caspases)/DIABLO (direct IAP-binding protein with low pI) in mammals and the RHG proteins Reaper, Hid, Grim and Sickle in fruitflies can remove the IAP-mediated negative regulation of caspases.
AIF, apoptosis-inducing factor; APAF1, apoptotic-protease-activating factor-1; Cyt c, cytochrome c; EndoG, endonuclease G; HTRA2, high-temperature-requirement protein A2.
Role of Caspases
• Effectors (cell disassembly) (caspases 2,3,6,7) and initiators (caspases 8,9)
• 14 identified mammalian caspases- 12 in humans
• Cysteine protease that has an absolute requirement requirement for cleavage after aspartic acid
• High specificity for which proteins are digested
– PARP (116 kDa) nuclear polymerase that repairs DNA is cleaved by caspase 7.
Types of Caspases
• Nedd2/Ich-1/caspase2• YAMA/CPP32/apopain/caspase-3- most characterized
• TX/Ich-2/ICErelII/caspase-4• TY/ICErelIII/caspase-5• Mch2/caspase-6• ICE-LAP-3/Mch-3/CMH1/caspase-7• FLICE/MACH/caspase-8-most proximally activated caspase
• ICE-LAP-6/caspase-9• Mch-4/FLICE 2/caspase-10• Ich3/caspase-11
Caspases (cysteine aspartic acid-specific proteases) are highly specific proteases that cleave their substrates after specific tetrapeptide motifs (P4-P3-P2-P1) where P1 is an Asp residue. The caspase family can be subdivided into initiators, which are able to auto-activate and initiate the proteolytic processing of other caspases, and effectors, which are activated by other caspase molecules. The effector caspases cleave the vast majority of substrates during apoptosis.All caspases have a similar domain structure comprising a pro-peptide followed by a large and a small subunit (see figure). The pro-peptide can be of variable length and, in the case of initiator caspases, can be used to recruit the enzyme to activation scaffolds such as the APAF1 apoptosome. Two distinct, but structurally related, pro-peptides have been identified; the caspase recruitment domain (CARD) and the death effector domain (DED), and these domains typically facilitate interaction with proteins that contain the same motifs. Caspase activation is usually initiated through proteolytic processing of the caspase between the large and small subunits to form a heterodimer. This processing event rearranges the caspase active site into the active conformation. Caspases typically function as heterotetramers, which are formed through dimerization of two caspase heterodimers. Initiator caspases exist as monomers in healthy cells, whereas effector caspases are present as pre-formed dimers.Not all mammalian caspases participate in apoptosis. For example, caspase-1, caspase-4, caspase-5 and caspase-12 are activated during innate immune responses and are involved in the regulation of inflammatory cytokine processing (for example, IL1 and IL18). Interestingly, caspase-12 is expressed as a truncated, catalytically inactive protein in most humans (caspase-12S*). However, a subset of individuals of African descent express full-length caspase-12 (caspase-12L*) and these individuals appear to be more susceptible to inflammatory diseases. To date, 400 substrates for the mammalian caspases have been identified, but the significance of many of these cleavage events remains obscure.
Caspase 3
• Caspase 3 (CPP32/apopain/YAMA)– shares similarity to CED 3.– Protein substrates include:
• PARP (poly ADP ribose) polymerase• PKC• sterol-regulatory element-binding protein• DNA dependent protein kinase (DNA repair)• U1-associated 70 kDa protein (mRNA splicing)• MEKK• DNA fragmentation factor- cytosolic factor that induces
nuclear fragmentation
• DNA damage-apoptosis model structure. The diagram depicts the general structure of the model. The model contains mathematical equations describing the individual protein-protein interactions and catalytic reactions for over 80 species that fall within the broad outlines shown. The equations are solved in specialised software including MatLab, and are run multiple times to simulate multi-cell environments.
p53 Dependent Apoptotic Pathways
Schematic representation of the p53-dependent apoptotic pathways by transcriptionalactivation of BAX, PUMA and APAF-1.