phgy 170 final- module 4 (2nd half)

Question 1

What are the structure and function of lipid kinases

Answer 1

• they are a class of signalling protein that phosphorylate phospholipids in the cytoplasmic leaflet of the membrane
• lipid kinases will add a phosphate to the polar head group and they phosphorylation of the head group results in a conformational change in the phospholipid and allows it to bind to its target protein in the membrane to pass the signal down the pathway
• some phospholipids can be phosphorylated more than once to become an active signal molecule

Question 2

what is the structure and function of adaptor proteins?

Answer 2

• nearly all signal transduction pathways have another class of proteins that are neither receptors or enzymes, and these are known as ADAPTORS
• these proteins have different binding domains that recognize phosphorylated amino acids of other “activated” structures on signalling proteins
• these domains along with others form the “glue” to hold elements of signalling networks together at the right time and place in a cell
• the adaptor proteins are important to allow cascades to be associated in the right space and time to fulfill their tasks and when and where they are needed in the cell

Question 3

what is an example of an adaptor protein.

Answer 3

• when the tyrosine of FGF-receptor is phosphorylated, the adaptor protein Grb2 binds, and undergoes a conformational change that allows it to bind to Sos
• Grb2 holds these elements of a signalling network together

Question 4

what are second messengers? What are their functions?

Answer 4

• the importance of proteins in signal transduction was emphasized, and we may have noticed that several non-protein ions or molecules are formed/ released such as cAMP calcium ions
• these non protein ions and molecules are called SECOND MESSENGERS
• they have this name because they relay signalling information from signalling proteins to other cellular targets
• the first messengers are the ligands that interact with the receptors

Question 5

what are the features of second messengers?

Answer 5

they are small in size, diffuse rapidly into the cytosol or membrane, amplify signals so that the interaction of just a few ligands with their receptors can trigger a much larger response within a cell by mobilizing second messengers, and they do not hang around in the cytosol for too long

• second messengers such as cAMP and cGMP are degraded by specific enzymes called phosphodiesterases, while ionic messengers such as Ca2+ are sequestered into cellular organelles
• other examples include small, hydrophobic molecules like diacylglycerol (DAG) and inositol triphosphate (IP3) and some gases including nitric oxide (NO)
• second messengers amplify signals to increase the response and this transduction feeds back to reception processes, and responses feed back to transduction, reception and the signal

Question 6

what are heterotrimeric G-protein signalling cascades, and what are the 4 main types?

Answer 6

• they regulate a multitude of signalling pathways in human cells
• the 4 main classifications of the signalling cascades include, GPCRs, cAMP, PKA and CREB

Question 7

what is the structure and function of GPCRs?

Answer 7

• there are over 800 different GPCRs that respond to a wide variety of stimuli, such as chemical modulators (ex. neurotransmitters, hormones and even odorants) and physical stimuli (ex. light)
• the signal transduction pathway is initiated by the bonding of a ligand to the GPCR
• binding of the receptor allows the receptor protein to interact with the heterotrimeric G-protein

Question 8

what is the structure and function of cAMP?

Answer 8

• the ligand-bound receptor stimulates the replacement of GDP for GTP in the G-α subunit which causes the heterotrimeric G-protein to dissociate from the receptor and itself to leave a G (β, γ) subunit, and an activated G-αs-GTP
• in this example, the Gαs-GTP then binds and activates the signalling protein adenylyl cyclase to convert ATP into cAMP, a second messenger

Question 9

what is the structure and function of PKA?

Answer 9

• next, cAMP can bind yet another signalling protein, protein kinase A (PKA)
• inactive PKA is a tetrameric protein with 2 regulatory subunits and 2 catalytic subunits
• the binding to cAMP to the regulatory subunits causes the protein to dissociate and release the active catalytic subunit
• once active, the catalytic subunit can phosphorylate a number of cellular proteins

Question 10

what is the structure and function of CREB?

Answer 10

• in this particular example, active PKA catalytic domains can enter the nucleus
• a common nuclear target is the cyclic AMP response element binding protein (CREB)
• once phosphorylated by PKA, CREB binds CBP (CREB binding protein) and together, the 2 proteins can interact with DNA to initiate transcription

Question 11

what are phospholipid kinase signalling cascades, and what are the 5 main types?

Answer 11

• phospholipid kinase is another protein involved in signalling cascades and there are GPCR, PLC, PIP2/IP3, Ca2+ and PKC

Question 12

what is the structure and function of GPCR?

Answer 12

• The signal transduction pathway is initiated by the binding of a ligand to the GPCR
  Binding of the receptor allows the receptor protein to interact with the heterotrimeric G-protein
  The ligand-bound receptor stimulates the replacement of GDP for GTP in the Gɑ subunit
  This causes the heterotrimeric G-protein to dissociate from the receptor and itself to leave a G(β,γ) subunit and an activated Gɑ-GTP

Question 13

what is the structure and function of PLC?

Answer 13

• In this example, the Gɑ-GTP then binds the phospholipid kinase signalling protein phospholipase C (PLC)

Question 14

what is the structure and function of PIP2/ IP3?

Answer 14

• Activated PLC breaks down the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to release 2 second messengers: diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3)

Question 15

what is the structure and function of Ca2+?

Answer 15

• IP3 diffuses freely in the cytosol and activates its receptor on the endoplasmic reticulum, which opens a ligand-gated calcium channel
  Ca2+ leaves the endoplasmic reticulum and, acting as a second messenger, can activate a number of calcium binding proteins (previously calmodulin has been used as an example

Question 16

what is the structure and function of PKC?

Answer 16

• Together, the membrane-bound diacylglycerol and the cytosolic Ca2+ bind to protein kinase C (PKC) resulting in its activation
  Activated PKC has numerous cellular targets that it can phosphorylate to modulate the target’s activity

Question 17

what are protein kinase signalling cascades?

Answer 17

• Protein kinases induce a cascade that results in the phosphorylation of several proteins within the cell and the 3 main types are FGFs, Grb2 and Erk

Question 18

what is the structure and function of FGFs?

Answer 18

• Fibroblast growth factors (FGFs) are a class of proteins that stimulate the growth of most mammalian cells
• FGFs bind to a family of receptor proteins called FGF receptors (FGFRs)

-FGFR os a homodimeric receptor kinase (tyrosine kinase)

-Binding of FGF to FGFR causes the subunits to dimerize (come together)

-Once bound together, the FGFR undergoes tyrosine transautophosphorylation to form phosphotyrosines on the cytoplasmic side

-These phosphotyrosines can be bound by a multitude of different proteins

Question 19

what is the structure and function of Grb2?

Answer 19

• Once such phosphotyrosine binding protein is the adaptor protein Grb2:

-Binding to a phosphotyrosine causes Grb2 to undergo a conformational change to bind Sos

-Sos activation leads to its binding to a monomeric G-protein Ras

-Binding of Sos to Ras replaces the GDP with GTP and the now active Ras can bind to a serine/threonine kinase called Raf

-Activated Raf can phosphorylate another protein kinase called MEK, which in turn will phosphorylate another serine/ threonine kinase named Erk

Question 20

what is the structure and function of Erk?

Answer 20

• Phosphorylated Erk forms a dimer and can phosphorylate other signalling proteins in either the cytosol or the nucleus

-In this example, Erk enters the nucleus to activate transcription factors, ultimately initiating transcription

Question 21

why does the cell need to break down molecules?

Answer 21

• many components of the cell need to be broken down or degraded as they are no longer needed or are damaged
• this may produce dangerous waste products that need to be disposed of carefully

Question 22

what are lysosomes, proteasomes and peroxisomes, and what are some of their basic functions?

Answer 22

• Lysosomes, proteasomes and peroxisomes handle dangerous cargo within the cell

—–> Lysosomes
- Lysosomes are organelles that break down misfolded and damaged organelles, nucleic acids, lipids and more

—–>Proteasomes
-Proteasomes are protein complexes that specifically break down damaged and misfolded proteins in the nucleus and cytosol

—–>Peroxisomes
-Handle dangerous free radicals including reactive oxygen species

-These are also problematic to the cell, and the cell needs a safe place to use these chemicals

Question 23

how does cargo get to the lysosome?

Answer 23

• Misfolded or non-functional proteins, as well as other cellular contents, are tagged for delivery to the lysosome
• Cargo is delivered to the lysosome in an endosome via the endomembrane system
• Cargo is targeted to the lysosome by a specific mannose-6-phosphate (M6P) sugar tag

-Interestingly, the enzymes that degrade these damaged proteins, called proteases, are also directed to the lysosome with the same M6P tag

—–>Vesicles
-The engulfed proteins including the membrane proteins and soluble proteins are delivered by the vesicles that empty their contents by fusing with the lysosome and are digested by the proteases

—–>Proteases
-The proteases are synthesized in the ER, tagged with M6P, and delivered to the lysosome by vesicles
They digest both soluble proteins and membrane proteins in the lysosome

Question 24

how does digestion in the lysosome occur?

Answer 24

• Lysosomes are mainly responsible for the breakdown of proteins that are not endogenous to the cell or from other organelles

-The lysosome contains high concentrations of proteases, which, as mentioned earlier, cleave both membrane proteins and proteins contained in the lysosome

-The lysosome also contains enzymes that cleave and digest fats and sugars, and can even engulf other organelles like damaged mitochondria or bacteria
Once large molecules have been broken down into their basic parts (like proteins to amino acids) they are transported to the cytosol so the cell can reuse them

Question 25

how does protein degradation by the proteasome?

Answer 25

- The proteasome degrades intracellular proteins (endogenous to the cell) in the cytosol and nucleus -The proteasome also requires the process of ubiquitination, another form of post-translational modification of proteins ----->Cytosolic proteins -Cytosolic proteins that have been misfolded or damaged are tagged with a polyubiquitin chain, which is composed of multiple molecules of ubiquitin - Ubiquitin: a small, regulatory protein that is attached to another protein which labels them for destruction by the proteasome -Multiple ubiquitins are required for the protein to be targeted and recognized by the proteasome and be degraded ----->Nuclear proteins -Proteasomes are also located in the nucleus so the cell can degrade unwanted nuclear proteins without having to export them to the cytosol -Damaged histones, for example, can by polyubiquitinated in the nucleus and then degraded by nuclear proteasomes

Question 26

what are the function of peroxisomes?

Answer 26

- Oxidizing agents like peroxides, ions, and free radicals are very hazardous to the cell -Peroxisomes serve as a place to keep and use these reactive oxygen species (ROS) safely using enzymes including catalase -Reactive oxygen species (ROS): These are highly reactive species formed from oxygen like superoxides and peroxide -Peroxisomes are small, membrane-enclosed organelles, and contain enzymes that catalyze a variety of metabolic reactions -Essential peroxisome proteins are called peroxins; they are synthesized in the cytosol and are targeted to the peroxisome by specific peroxisomal targeting signals (PTSs) -Although they are hazardous, peroxisomes also carry out important decomposing functions for some cargo such as uric acid, amino acids, and long chain fatty acids

Question 27

what are the two main forms of cellular death?

Answer 27

apoptosis and necrosis

Question 28

what is apoptosis?

Answer 28

-Apoptosis is referred to as programmed cell death and it is an energy-consuming process that cleanly and carefully ends the life of a cell -Apoptosis is used to protect the body from damaged cells that no longer function properly It is also used in development, for example, to remove the webbing between fingers and toes during fetal development

Question 29

what are the 4 main steps of apoptosis?

Answer 29

-----> INITIATION - Apoptosis is initiated by 2 different pathways: intrinsic and extrinsic - The cell initiates apoptosis itself - The intrinsic pathway originates in the outer membrane of the mitochondria and intracellular signals such as severe DNA damage, ROS, toxins, or other trauma will turn on the intrinsic pathway in the cell - External signals initiate apoptosis in the cell - The extrinsic pathway uses a plasma membrane receptor called the death receptor - Neighbouring cells, such as immune cells, will release death ligands, which bind to the death receptors on a damaged cell which then activates additional signals which lead to apoptosis -----> MEMBRANE BLEBBING AND ENZYME ACTIVATION - The cell begins the shrink and form blebs - Blebs: small protrusions from the plasma membrane - This is the first visible signal that a cell is undergoing apoptosis - Enzymes termed caspases (cysteine aspartate-specific proteases) are activated - The initiator caspases are activated by either the extrinsic pathway or intrinsic pathway - These caspases will cleave and therefore activate other caspases known as executioner caspases -----> CELL STRUCTURE CHANGES - After the executioner caspases are activated, the cell changes structure - DNA is fragmented, often between histones, and DNA repair halts - The nuclear membrane breaks down and the nucleus disappears - The cytoskeleton is also disassembled and the plasma membrane phospholipid content changes with scramblases, with PS (phosphatidylserine) being exposed on the exoplasmic leaflet of the plasma membrane - Organelles persist, and are enclosed in apoptotic bodies -----> ENGULFMENT - Phagocytes endocytose the apoptotic bodies to dispose of them - These are then safely digested by the phagocytes’ lysosomes - This causes a minimal amount of disturbance to the cells and surrounding tissue

Question 30

what is necrosis?

Answer 30

- Unlike apoptosis, necrosis is normally the result of the cellular injury - When injured, cells will attempt to repair themselves - These stressed cells may show swelling in the mitochondria and endoplasmic reticulum or some blebbing, but will return to normal if they are able to recover - Necrosis is the major pathway of cell death as a result of severe damage that is not able to be repaired The cell’s organelles are not able to function and it dies

Question 31

what are the 4 main steps of necrosis?

Answer 31

-----> DAMAGE - The cell is damaged beyond repair - There can be numerous causes for necrosis including TOXINS: sources include bacterial infection, drugs, chemicals, etc EXTREME HEAT OR RADIATION: proteins denature, DNA is damaged FREEZING: ice crystals puncture the cell membranes and organelles ISCHEMIA: blood flow is stopped to the tissue; lack of oxygen, glucose, etc.., prevents the cell from receiving the necessities of life PATHOGENS: bacterial or fungal infections MECHANICAL TRAUMA: physical injury to the cell -----> SWELLING - The organelles begin to lose their structures and swell Vacuoles, or undefined bodies, form in the cell Depending on the type of damage, the DNA may be degraded -----> DESTRUCTION - The cell membrane and remaining organelles lose structural integrity - Holes can be observed using microscopy - The cellular content spills out of the cell, producing inflammatory signals - The mitochondria’s proteins are released, and lysosomal contents are exposed - Cells nearby are exposed to these remains of the cell, and are also damaged or have apoptosis signaling triggered - Unlike apoptosis, it is difficult for the body to clean up the cellular remains after necrosis

Question 32

differences between necrosis and apoptosis

Answer 32

-----> CAUSES - in APOPTOSIS, DNA damage, withdrawal of essential growth factors or nutrients, detachment from substrate, attack by cytotoxic lymphocytes - in NECROSIS, trauma is what induces the DNA damage -----> KEY FEATURES - in APOPTOSIS, there are nucleus fragments, cell shrinkage, cell fragments, and apoptotic bodies - in NECROSIS, the cell swells, cell bursts, organelles break down, and dangerous compounds form -----> END RESULTS - in APOPTOSIS, there is engulfment of fragments and no inflammatory response - in NECROSIS, there is an inflammatory response