1
Q
MHC 1: what they bind
MHC 2: what they bind
A
- MHC 1: bind peptides derived from proteins made in the cell itself
- MHC 2: bind and display peptides from protein that has been phagocytized
2
Q
Purpose of MHC Molecules
A
- allow certain cells of the immune system to examine them via T cell receptors
3
Q
MHC 1: Structure
A
- two polypeptide chains
- first is long and consists of an intracellular domain, a transmembrane domain, and three extracellular domains
- second polypeptide chain is short and consists of one domain
4
Q
What should you notice?
A
Notice:
- peptide nestled in the top of the molecule
- in this context that the TCR receptor binds its specific peptide antigen
- Domains based on beta sheets
- homologous with the domains from which antibodies and T cell receptors are built
- alpha helices that create the groove in which the peptide is bound
5
Q
Steps from protein in cell to being expressed in MHC I molecule on the surface of the cell.
A
- Marked for destruction by ubiquitin
- Ubiquitin ligases have the inherent ability to recognize abnormal proteins
- Virally infected/misfolded protein
- Ubiquitin ligases have the inherent ability to recognize abnormal proteins
- Protein degraded to peptides by proteosome
- typically 9 amino acids
- peptides are transferred into the rough ER via a TAP transporter
- Meanwhile, an MHC I molecule is synthesized and placed in the membrane of the rough ER
- peptide binds in the groove in an MHC I molecule
- combination moves through the Golgi apparatus and into a secretion vesicle
- Exocytosis of the secretion vesicle places the MHC I molecule with its peptide on the surface of the cell
- peptide and MHC I molecule are now in position to be recognized by a T cell receptor on a T cell
6
Q
MHC II: Structure
A
- also has two polypeptide chains
- each polypeptide chain consists of an intracellular domain, a transmembrane domain, and two extracellular domains
- domain structure is similar to the MHC I molecule.
- big difference, however, is that a peptide from a phagocytized protein is bound the the MHC II molecule on the surface of the cell
7
Q
Steps in phagocystosed protein being expressed on MHC II molecule
A
- pathogen is phagocytized, winding up in a phagocytic vesicle
- lysosome with proteases fuses with the phagocytic vesicle, and the proteases digest the proteins into peptides
- Meanwhile the MHC II molecule is synthesized in the rough ER
- vesicle with the MHC II molecule now fuses with the vesicle containing the peptides, and a peptide bind to each MHC II molecule
- Exocytosis again places the MHC molecule and its peptide on the surface of the cell
- peptide and MHC II molecule are now in position to be recognized by a T cell receptor on a T cell
8
Q
Do you remember the term for the general type of molecule used by phagocytes to recognize a newly encountered foreign molecule? In other words, what is the general type of molecule used to recognize an antigen before a specific immune response has had a chance to make antibodies or T cell receptors?
A
A good term is innate receptors. One important group is the toll-like receptors. Another is the mannose receptor, which recognizes a repeating carbohydrate pattern
9
Q
Do you recall the term for the type of molecule that specifically binds the peptide displayed on an MHC molecule?
A
T cell receptor
10
Q
Dendritic Cells: Role and Action
A
- distributed throughout the body
- phagocytosis is their key process
- not primarily for the purpose of destroying microbes
- capture antigens and transport them to lymphoid tissue
- facilitate the development of an immune response
- Display peptides in MHC II molecule
11
Q
Helper T-Cell: Role and Action with dendritic cells
A
- In lymphoid tissue
- check to see if their T cell receptors specifically bind the peptide displayed in the MHC II molecule on dendritic cell
- certain dendritic cells also have a mechanism for displaying peptides on MHC I molecules
12
Q
Two Common Features of T-Cells
A
- All T cells have T cell receptors
- remain attached to the membranes of the T cells
- always recognize peptide antigens presented on MHC molecules
*
13
Q
CD4+ T-Cells
A
- bind peptides displayed on MHC II molecules
- Only with phagocytic cells
- dendritic cells (or macrophages)
- B cells
14
Q
Activated T-Cells
A
- T-Helper Cells
- T cell recognizes its specific peptide antigen presented on a dendritic cell or B cell
15
Q
TH1 vs. TH2 Cells: Tendency to Form
A
TH1 tend to form when:
- lots of strong stimulation by the phagocytized antigen
- lots of activation of the innate immune system
TH2 tend to form when:
- weaker, more prolonged stimulation
- less activation of innate mechanisms
16
Q
Important Role of TH1
A
- travel around the body to macrophages that have phagocytized the antigen
- bind and release IFN-gamma
- increases the fusion of lysosomes with phagosomes
17
Q
Why aren’t macrophages always active?
A
- Killing mechanisms can damage the body as well
18
Q
Which of the following do you suppose is treated sometimes with IFN-gamma?
a. hepatitis A
b. hepatitis B
c. multiple sclerosis
d. chronic granulomatous disease
e. rheumatoid arthritis
A
D: You have got it! The interferon-gamma would be expected to stimulate exactly the process that is weak in macrophages in this disorder.
19
Q
Helper T Cells and “helping B cells”
A
- Help B cell’s respond to antigens
- Many antigens cannot by themselves cause a specific B cell to divide into a clone of antibody secreting plasma cells
- B Cell Divide into Clone:
- Phagocytose antigen and display peptides on MHC II
- activated helper T must bind to the cell
- T helper release appropriate cytokines
20
Q
TH1 vs TH2: Types of antibodies made
A
- cytokines secreted are different for different types of helper T cells
- TH1
- Heavy chain switching to make IgG
- Good opsonin
- TH2
- IgE
- IgM
- IgA (MALT)
21
Q
what term refers to the type of antigen that can activate a B cell without necessarily requiring help from helper T cells?
A
multivalent
22
Q
When do CD8+ T-Cell Divide into Clone?
A
- following its encounter with another cell displaying its specific peptide on an MHC I molecule
- In a lymph node this initial cell is likely to be a dendritic cell
- Termed Cytotoxic T-Cell
23
Q
Cytotoxic T-Cells
A
- look for ordinary cells in the body displaying the specific peptide on MHC I molecules
- Virally infected cells
- Must undergo apoptosis
24
Q
How Cytotoxic T-Cells Induce Apoptosis
A
- TCR to the peptide in the MHC I
- forms an adhesion complex with the infected cell
- Releases secretion vessicles
- Secretion vessicles contain perforin
- forms channels in the infected cell
- Also contain granzyme
- proteases that activate certain caspases
- Set of enzymes that trigger apoptosis processes
- proteases that activate certain caspases
- Fas ligand, can also activate caspases via another pathway
25
From lecture, what are some of the things that happen during apoptosis?
1. The cell tends to detach from whatever it is normally attached to.
2. The mitochondria become permeable, releasing their contents into the cytosol.
3. The nucleus breaks down, releasing its contents. Nucleases break apart both the cellular and viral nucleic acids.
4. Phospholipids in the plasma membrane tend to evert, so that the outside of the cell now has unusual phospholipids on the outside.
5. The plasma membrane become permeable and the influx of fluid gives the cell its blebbed appearance.
26
Most Effective Opsonins
* antibodies of the type IgG and IgM
* antibody binds to its antigen on a microbe
* Fc region of the antibody binds to an Fc receptor on a neutrophil or macrophage
* Phagocytosis
27
Chronic Infections
* Phagocytosis by itself may not be enough to kill the microbes engulfed
* Ex: TB: lysosomes are prevented from fusing with the phagosome
28
What Happens In Macrophage Activated State?
* more of the killing factors are synthesized, and the lysosomes are more likely to fuse with the phagosome
* more potent mix is released into the phagolysosome.
29
Complement System
* can be activated via antibodies bound to microbes
* either IgG or IgM, especially the pentameric form
30
Which one of the following best describes how a TH1 cell identifies a macrophage to activate?
a. By binding to the macrophage using molecules of innate immunity
b. By its T cell receptor binding to peptide antigen displayed on MHC II molecules
c. By its T cell receptor binding to peptide antigen displayed on MHC I molecules
d. By binding to the macrophage with C3b attached.
e. By the macrophage using a Fc receptor to bind to IgG.
B
Good! Helper T cells are CD4+, which means that bind to antigen displayed on MHC II molecules.
31
Eosinophils
* have an Fc receptor for IgE
* IgE binds to the cuticle of a helminth
* Fc receptor on an eosinophil can bind to the Fc region on the IgE
32
Mast Cells
* found especially under epithelia
* large secretion vesicles filled with paracrine secretions like histamine
* mast cells have Fc receptors for IgE
* multivalent antigen appears again, it causes the the Fc receptors to cluster
* receptors phosphorylate each other
* tyrosine kinase
* exocytosis of the vesicles and synthesis of prostaglandins and leukotrienes
33
Mucosal Immunity
* IgA is joined into dimers with a J-chain
* binds to the basal surface of the intestinal or respiratory epithelium
* engulfed by the cell, transported to the luminal membrane, and released into the lumen
* dimeric IgA binds to bacteria or viruses
* prevent their binding to the epithelium
* cause agglutination
* class of lymphocytes found within the epithelium itself
* largely T cells
* majority of the CD8+ variety
* respond to some of the more common microbes in the lumen
34
Mucosal Membranes
* secretions such as saliva
* antibacterial enzyme, lysozyme
* antimicrobial peptides such as defensins
* IgA
* dimeric form binds to the inner surface of epithelial cells
* engulfed by the cell by endocytosis
* moves in a vesicle across the cell and is released into the lumen by exocytosis
* IgA prevents binding and causes agglutination, swept away by mucous/body
* epithelium also has lymphocytes embedded in it
* special population that respond mainly to a restricted set of antigens typically found in the lumen of the GI tract and respiratory airways
* Most are CD8+ T cells that kill infected cells
35
Pathogens in Interstitial Spaces, Lymph and Blood
* phagocytosis by neutrophils or macrophages is its most likely fate
* most effect opsonin is antibody in the form of IgG (or IgM)
* Agglutination by these antibodies helps phagocytosis
* Complement System
* innate, IgG, especially IgM pentamer
* opsonin C3b, MAC
* C3a and C5a peptides serve as inflammatory paracrines
* helminth worms, eosinophils become involved, especially if IgE is made
* TH2 helper T cells
* Eosinophils have vessicles with basic and toxic proteins
* myeloperoxidase, oxygen radical story
* Mast cells also release inflammatory paracrines
36
Pathogens in the Cytosol of Cells
* slow the spread of the infection with IFN-alpha and IFN-beta
* induce in nearby cells an "anti-viral state"
* synthesis of certain proteins that slow viral replication and slow down cell growth and division
* ultimate solution is apoptosis
* caused by natural killer cells of the innnate system and cytotoxic T cells of the specific immune system
* Macrophages then engulf the remains
*
37
Pathogens in vessicles inside cells
* "activation" of macrophages by **TH1 helper** T cells is the next step
* recognizes the macrophage due to antigen displayed on an MHC II molecule
* helper T cell releases** IFN-gamma**
* increases the fusion of lysosomes with the phagosome and increases synthesis of oxygen radicals and other killing mechanisms
* Cytokines are also released that cause inflammation and recruit further cells
* wall off the pathogen through the formation of a **granuloma**.
38
Table summarizing effector mechanisms and locations
39
Ordinary Influenza Infection
The virus first binds to and infects cells of the upper airways:
* macrophages phagocytize virus and release cytokines such as TNF-alpha, etc.
* natural killer cells slow the infection
* cytotoxic T cells (CTL) cause virally infected cells to undergo apoptosis
* IgG promotes phagocytosis of any viruses entering the interstitial fluid
* IgA is transfered to the lumen of the airways in order to bind to the viruses and prevent them from attaching to the epithelial cells
40
Secondary Bacterial Infection
* person with a compromised immune system, it can lead to **bacterial pneumonia**
* lung tissue fluid filled at autopsy
* edema, neutrophils and other white blood cells fill the delicate alveoli
41
Hemagglutinin and Neuraminidase
* Influenza A has two important surface molecules
* **hemagglutinin**: binds to the surface of cells in the body, following which the cell takes up the virus and become infected
* **neuraminidase**: enzyme that cleaves the molecule on the host cell that binds hemagglutinin. Hemagglutinin attachment must be broken for the new viruses to be free to move on and infect other cells.
42
Hemagglutinin and Neuraminidase Types
* 16 different hemagglutinin types and 9 different neuraminidase
* Antibodies against one type not effective against another type
43
Influenza A
* Bird Virus
* Affect human: H1N1, H1N2 and H3N2
* H5N1 not normally infect humans
* but when does, deadly
* H7N9 also deadly
* doesn't move from person to person well
44
Antigenic Drift
* small variation, which creates a new strain
* hemagglutinin and/or neuraminidase molecules acquire **point mutations**
* This is why flu appear regularly
* Requires a new vaccine
* Create **epidemics** of flus
45
Antigenic Shift
* Causes **pandemics **
* a new **subtype** is created
* Flu A: genome is encoded in eight separate segments of RNA
* **two different subtypes of influenza A infect the same individual**, it is possible for new viruses to wind up with segments of RNA from both sources
* Usually pig/bird with double infection
* antigenic shift occur in H5N1 or H7N9 to make human-human easy, cause a deadly pandemic
* Influenza B cannot undergo antigenic shift
46
Influenza Vaccines and Antiviral Medications
* three subtypes that the WHO feels are most important for the year
* This years: protects against an influenza A H3N2 virus from 2012, the H1N1 virus that emerged in 2009 to cause a pandemic, and an influenza B virus
* Intranasal flu vaccine: attenuated, live viruses, which usually work by causing mucosal immunity
* Antivirals against influenza A
* **neuraminidase inhibitors**
* Oseltamivir (Tamiflu) is taken orally
47
H5N1, H7N9, H1N1
* "Bird Flu"
* 2010, for example, WHO identified 40 cases of H5N1, of which 50% were fatal
* H7N9 is fairly similar
* H1N1 subtype known as "swine flu"
* When leads to hospitalization, significant portion of people die
* Dangerous forms that can move deep into lungs and cause** viral pneumonia**
*
48
Bacterial vs. viral pneumonia
* Bacterial: virus infects the upper airways
* secondarily cause bacteria to move into lungs
* fluid and neutrophils in aveoli
* Viral: directly infect epithelium lining aveoli
* fluid and cells accumulate in interstital space
* necrosis
49
What causes flu infection to be fatal?
* H5N1 causes considerable release of **TNF-alpha**
* cytokine storm
* **Septic Shock** (sepsis: bacteria and toxins, septicemia: only bacteria)
* endothelial permability increase, not enough blood profusion to tissues
* **Disseminated Intravascular Coagulation (DIC)**
* clotting factors and platelets depleated
* organ failure due to clots
* TNF-alpha and IL-1, make clots form
* **Acute Respiratory Distress Syndrome (ARDS)**
* epithelial inflammation out of hand
* dyspnea and tachypnea
* once develop, mortality 50%
50
Given the seriousness of a potential influenza A (H5N1) pandemic, why aren't we being vaccinated right now?
The antigenic shift/drift has not taken place yet. Cannot create vaccine for an unknown virus
51
Hepatitis
* Hep A: acute disorder
* liver cells infected and macrophage/neutrophils cannot get at them
* IFN-alpha and IFN-beta are released by virally infected cells
* Induce an anti-viral state
* Number of processes that lead to release of interferons.
* Toll-Like Receptor and cytosolic protein that responds to RNA
* NKC cause apoptosis of the infected cells
* In one to two weeks, make antibodies, igM and IgG
* cytotoxic T cell: causes the flu like symptoms
* Hep B and C: immune system not good enough to clear out,
* Chronic
* treated with IFN-alpha
* Increases the MHC1 molecules on cell surface
52
Tuberculosis
* dendritic cells phagocytosis some of virus
* TH1 helper cells
* lots of antigen and strong stimulation by antigen
* Bind MHC II of macrophage
* IFN-gamma
* greater fusion of the lysosomes with phagosomes, phagocyte oxidase, and the release of cytokines that cause inflammation and recruit further macrophages.
* If not work, additional macrophages, leading to a granuloma
* Bacteria walled off but still tissue damage because helper T cells strongly stimulate the killing mechanisms and promote strong local inflammation
* Granuloma can come back as secondary TB
53
Anthrax
* Taken by dendritic cells to lymph node
* Become vegitative and start dividing
* **Capsules** inhibit phagocytosis
* causes hemorrhagic **lymphadenitis**, enter blood in large numbers
* Toxins released by bacteria, **systemic inflammatory response**
* Release oxygen radicals and TNF-alpha and IL-1
54
Ebola
* Enters through mucous membrane or break in skin
* macrophages and dendritic cells infected
* virus **suppresses type I interferon** and divides rapidly
* hypotension and shock
* macrophages synthesis tissue factor and d dimer becomes present in blood
* After three or four days, platlets are low, and bleeding,
* **hemorrhagic fevers**
55
What type of lymphocyte without T cell receptors causes apoptosis of virally infected cells?
Natural Killer Cells
56
Patient B
* On day 10 hematocrit is elevated
* Dehydration concentrates the blood
* Collapsed inferior vena cava
* Dehydrated, hypovolemia
* Edema in intestinal walls
* Increased permeability of endothelial
* High C-Reactive protein
* Lots of TNF-alpha and IL-1
* Day 14 or 15
* More WBC and CRP
* Less viral RNA and less diarrhea
* Secondary bacterial infection from crossing compromised intestinal mucosa
* Elevated lactate shows signs of septic shock, mitochondria not recieving enough oxygen
* Epistaxis (nose bleeding) because of thrombocytopenia from platelets in endothelial
* D-dimer levels show fibrin being formed throughout body, DIC
* Increased breathing to offset lactic acidosis
57
Atopic Response (Definition)
* Type I hypersensitivity
* Immediate hypersensitivity
* Allergies
58
Sequence in Atopic Response
* Antigen called allergen
* Inappropriate formation of TH2 cells
* Small antigens with little inflamation
* Steer towards IgE formation
* Binds Fc receptors on mast cells
* Within an hour
* Secretion of mast cell vesicles
* histamine
* Release eicosanoids
* Leukotriene C4 (anti-eicosanoid and LT drugs)
* Begins in about 2-4 hours
* Cytokine secretion from mast cells
* Notable, TNF-alpha
* Recruits more WBC (TH2, eosinophils)
* Eosinophils release their toxic proteins
* Long-term chronic inflammation
* Treat with glucocorticoids (corticosteriods)
*
59
Anaphalaxis
* systemic response to an allergen
* can lead to shock
* uticaria in skin
* mucosal irritation leading to vomiting and diarrhea
* airways swell making it hard to breathe
* penicillin=hapten (reponse after binding to protein in body)
* Epinephrine to raise blood pressure and relax airways
60
CD4+ T cell Activated Macrophages in "Delayed Allergic" Responses
* Involves Tcells and activated macrophages
* Second introduction of antigen produces a larger response
* macrophages producing damage
* Contact dermatitis (hapten)
* Langerhans in epidermid carry to lymph node
61
Autoimmune Diseases Due to Antibodies
* B-Cells making antibodies
* myasthenia gravis, antibodies bind to and inactivate acetylcholine receptors on skeletal muscle
* IgG or IgM
*
62
autoimmune thrombocytopenia
Antibodies bind platelets and lead to destruction
63
Graves disease
* B-cells secrete antibody that binds to a protein
* Stimulating antibody bind to TSH receptor
* Release thyroid hormone
* Goiter
64
Rheumatic fever
* antibodies produced to act on a pathogen
* streptococci cross-react with molecules in the body
* Damage to heart muscle and valves
65
systemic lupus erythematosus (SLE)
* **complexes of the antibody and antigen**
* young women
* makes antibodies against a number of molecules in the nucleus
* Complexes of the antibody and antigen form and cause damage as they are deposited in tissues
* glomerulus of the kidney
* skin and joints
* sun exposure leading to rash
66
Autoimmune Disorders Involving T cells
* T cells release cytokines that can cause inflammation directly or activate macrophages
* **Inflammation** is a central feature of these disorders and fits the chronic type pattern
* blocking TNF-alpha is an effective treatment
*
67
Antigen Spreading
* additional self-antigens become involved in the response
* as attacked, more self antigens exposed, to which the immune system might respond
68
In addition to cytokines, such as TNF-alpha, what are some of the factors that activated macrophages release?
enzymes, growth factors, oxygen radicals, NO
69
rheumatoid arthritis
* immune system attacks the synovial membrane
* synovial inflammation and destruction of joint
* early, bilateral stiffness
* nodules on bony areas where lots of movement
* synovial membrane inflammed
* **hyperplasia** and creates finger like extensions
* angiogenesis leading to abnormal structure, **pannus**
* destroys the joint
* pannus may extend over destroyed joint, leading to **ankylosis**
70
One treatment for rheumatoid arthritis is to inject antibodies that bind to a cytokine, thereby removing its action. Based on what you know, what might be a logical cytokine to attack in this way?
TNF-alpha or IL-1
71
One risk factor for rheumatoid arthritis is elevated C-reactive protein. Can you trace the sequence of events that would explain this?
TNF-alpha or IL-1 from macrophages enters the blood and stimulates release of acute phase proteins such as C-reactive protein. This is often associated with increased inflammation in the body.
72
multiple sclerosis
* immune system responds to proteins in the myelin sheath of nerve cells
* demylenation (especially visual pathways)
* double vision, weakness, paralysis and various other neurological symptoms, interspersed with periods of remission
*
73
type I diabetes mellitus
* insulin secreting cells attacked
* TH1 helper T cells and cytotoxic T cells are largely responsible
* Perhaps some autoantibodies
74
What is the medical term for "hives"?
urticaria
75
Membrane Potential
* **electrical potential difference **between the inside of the cell and the surrounding extracellular fluid
* especially important in muscle and nerve cells
* **changes in their membrane potentials** are used to code and transmit information.
76
What is an electrical potential difference?
* net separation of charge between the two locations
* measured in units of volts
77
Resting Membrane Potential
* typical neuron, this is about –70 millivolts
* minus sign indicates that the inside of the cell is negative
* **very small number** of negative and positive ions need to be separated by the membrane
*
78
energetic factors influence the movement of an ion across a membrane
The concentration gradient
* The electrical potential difference
* **Total energy change** for the movement of an ion across the membrane is the the **sum** of the energy change due to the **concentration gradient** and the energy change due to **electrical potential difference**
* May act in same or opposite directions
*
79
Depolarization and Hyperpolarization
* Depolarization: membrane potential to become **less negative**
* Hyperpolarization: become **more negative**
80
Diffusion
* hydrophobic layer creates a barrier
* Exception like NO and CO2 and nonpolar substance (hormones and fatty acids)
*
81
Channels
* multiple subunits form a pore
* Each subunit consists of multiple transmembrane domains
* Aquoporins and ion channels
* Factors affecting rate of flow through an open ion channel
* membrane potential
* concentration gradient
* Note that ions moving through an open ion channel affect the membrane potential.
82
Properties of Ion Channels
* Selectivity
* which ion allowed to travel through channel
* Gating
* open or closed
83
Ungated
* open all the time
* some K+ and Cl-
* Ca++ and and Na+ are never ungated
84
Voltage Gated
* open/close in response to membrane potential
* Key in generation of electrical signals in nerve, muscle, and cardiac cells
* **voltage-gated Na+ channel**
85
Ligand Gated
* opened when regulatory molecules bind
* neurotransmitter receptors are ligand gated channels
* example is the **nicotinic acetylcholine receptor**
* neuromuscular junction on skeletal muscle cells, and also at synapses in autonomic ganglia
86
Mechanically Gated
* Afferent neurons (sensory neurons)
* skin, touch and pressure
* auditory and vestibular
*
87
Temperature Gated
* Normally gated by temperature
* Some ligands can open them
* capsaicin for heat
* menthol for cool
88
Facilitated Diffusion
* carrier protein with specific binding site for molecule
* **Glucose Transporter**
* **Glucose Uptake**
* **specific binding site** for glucose
* **Concentration gradien**t determines rate of uptake
* **Does not require ATP**
* Control transport by controlled number of transport proteins in the membrane
* Low insulin, few glucose transporters
* High insulin, more glucose transporters
89
Coupled Transport
* two substances are required to bind in order for transport to occur
* Free energy driving the transport is the **sum of the free energies for transport of both substances**
* If it is in the same direction, it is called **cotransport**
* If it is in the opposite direction it is called **countertransport**
90
glucose across the apical plasma membrane of epithelial cells in intestinal cells
* sodium-glucose cotransporter
* carbobhydrate gradient will fluctulate
* always steep gradient favoring Na+ into the cell
* because of the sodium-potassium pump
* Na+ gradient can move glucose against it's concentration gradient
91
Saturation
* facilitated diffusion and coupled transport show **saturation**
* **transport maximum (Vmax)**
* every transporter is bound by substance
* transport rate cannot increase
92
Active Transport
* energy for transport is derived from ATP hydrolysis
* move molecules in one direction regardless of the concentration gradient
* **Na+/K+-ATPase**, or sodium-potassium pump
* moves three Na+ ions out of the cell and two K+ ions into the cell
* Consumes about a third of ATP at rest
* **Ca++-ATPases** and the **H+/K+-ATPase**.
* keeps the Ca++ concentration low in the cytosol
* Stomach acid secretion
*
93
ABC Transporters
* that depend upon ATP binding for transport
* **ATP-Binding Cassette**
* two nucleotide binding domains that bind ATP
* Ex: **multidrug resistance protein (MDR1)**
* pump a wide variety of nonpolar drugs and toxins out of cells
* resistance to chemotherapy in cancer cells
* Ex: **CFTR**
* forms a **Cl- channel** that is expressed on the apical plasma membrane of many epithelial cells.
* Defective in cystic fibrosis
* decreased secretion
* ion channel that is **regulated by both phosphorylation and ATP binding**
* bacterial toxins causing unregulated activity can lead to diarrhea
94
Amino Acids in the membrane
* Nonpolar sidechains face outward
* Polar peptide bonds face inward
95
Voltage Gated Sodium Channels: Organization
* 24 transmembrane domains
* 4 clusters of 6 TMD
* Each group of 4 is a homologous domain
* Clustered to form a channel in the middle
96
Voltage Gated Sodium Channel: Ion Selectivity
* Very selective for Na
97
Voltage Gated Sodium Channel: **gate**
* gate opens in response to regulatory molecules that specifically bind to either the inside or outside
* gate is controlled by a **voltage sensor**, which responds to the level of the **membrane potential**.
* **several voltage sensors** must respond before the gate opens
* **inactivation gate** limits the period of time the channel remains open
98
Voltage Gated Sodium Channel: During changes in membrane potential
* Each homologous domain has one transmembrane domain **a positively charged amino acid** is found at **every third position**, giving a **total of four to eight positive charges per transmembrane domain**.
* Voltage sensors
* Deoplarize, voltage sensor move towards outside and gate opens
99
Know these parts of intesine
* Lumen
* ECF
* Tight Junctions
* Apical membrane
* Basolateral membrane
* microvilli
* different sets of transporters are localized in either the apical or basolateral membranes
100
Epithelial: Absorption
* Glucose across the apical membrane by sodium-glucose cotransporter
* Na+ gradient is established by the **Na+/K+-ATPase**
* located on basolateral membrane
* Facilitated diffusion of glucose into the ECF is a **passive** process
* **glucose transporter**
101
Oral Rehydration Therapy
* Expand ECF
* requires getting Na+ absorbed into the body, since the amount of Na+ in the ECF is the major determinant of ECF volume
* glucose included to speed uptake of Na+
102
Epithelial: Secretion
* solutes are moved across the epithelium, which then draw water into the lumen by **osmosis**
* rate-limiting and regulated step in intestinal secretion is the movement of Cl- ions across the apical plasma membrane
* Cl- is transported into the epithelial cell by a cotransporter expressed on the basolateral membrane
* Na+ gradient, established by the Na+/K+-ATPase
* Cl- down concentration gradient via Cl- channel CFTR
103
CFTR
* member of the ATP-binding cassette (ABC) protein family
* ATP binding is used to open an ion channel
* **regulatory domain** that is phosphorylated by **protein kinase A (PKA)**, also known as **cAMP-dependent kinase**
* **alpha subunit of the G-protein** to activate the enzyme **adenylyl cyclase**
* Adenylyl cyclase produces the **second messenger cAMP** which activates PKA to phosphorylate CFTR
* **cholera** toxin chemically modifies the alpha subunit of the G-protein so that it cannot inactivate
104
Neuron: Cell Signaling Distance
* connected to their target cells via **synapses**
* A **neurotransmitter** crossing the small space in the synapse only travels about 20 nanometers
105
Norepinephrine Cell Signaling Distance Example
* **norepinephrine **is a neurotransmitter
* acts as a hormone when released from the **adrenal medulla**
106
Timing of Signaling
* neurotransmitters bind to receptors that are **ligand-gated** **ion channels**
* closes channel, changed membrane potential
* Neuronal signaling less than a second
* Paracrine/Endocrine signaling minutes to hours
107
Second Messengers
* intracellular signaling molecules
* especially true for receptors coupled to trimeric G-proteins
* **G-protein coupled receptors **or **GPCR**s
* example is the enzyme adenylyl cyclase
* uses ATP as a substrate to synthesize the second messenger **cyclic AMP** (**cAMP**)
* **small** and **diffusible**
* **amplify **a signal
* Ex: each active adenylyl cyclase is capable of generating many molecules of cAMP.
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Calcium
* 2nd messanger because cellular concentrations are kept very low
* **exocytosis of secretory vesicles**
* **regulation of contraction**
* One source of Ca++ is from the ECF
* ER, or the sarcoplasmic reticulum in muscle cells
* Ca++ released by second messanger **inositol triphosphate (IP3)**
* binds to ligand-gated Ca++ channels
* signal is rapidly terminated by **Ca++-ATPases**
* transport Ca++ out of the cytosol
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Calcium binding protiens
* Ca++ regulates cellular processes by binding to Ca++-binding proteins
* Ca++-binding protein that regulates skeletal muscle contraction is called **troponin**
* Ca++-binding protein found in all cells is the protein **calmodulin**
* Ca++/calmodulin does not perform a cellular process itself, rather it works by binding and activating other protein
* protein kinases
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GPCR: ligands, structure
* Ligands:
* peptide hormones, neurotransmitters, and odor molecules
* seven transmembrane domains
* many GPCRs have been identified in the human genome
* known as orphan receptors, because their ligands have not been identified.
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GPCR Activation
* GPCR associate with trimeric G-proteins
* alpha, beta, and gamma subunits
* Exchange GDP for GTP at alpha
* Dissociates into alpha and beta-gamma
* Alpha diffuse along membrane and activate enzymes to make second messangers
* Beta-Gamma diffuse along membrane and activate ion channels
* Inactive by alpha-subunit intrinsic GTPase activity
* With GDP, can reassociate with the beta-gamma complex
* **Cholera toxin** causes a chemical modification that prevents GTP hydrolysis and leads to unregulated signaling
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Adenylyl Cyclase
* catalyzes the formation of the second messenger cyclic AMP (cAMP)
* generate many molecules of **cAMP**
* amplify the signal
* bind to and activate **protein kinase A**
* PKA then phosphorylates target proteins in the cell
* cAMP is rapidly broken down by **phosphodiesterases**
* Cholera toxin
* destroys GTPase activity of G-alpha
* persistant adenylyl cyclase activity
* continuous cAMP
* continuous activation PKA
* Continuous phosphorylation of CFTR
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Phospholipase C
* cleaves PIP2
* generate two second messengers, **IP3** and **diacylglycerol (DAG)**
* IP3 (water soluable) binds and open ligand-gated Ca++ channel
* DAG (lipid soluable) activates protein kinase C (PKC)
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Desensitization
* G-protein Receptor Kinase (GRK)
* Phosphorylates on GPCR
* Beta-arrestin binds and prevents trimeric G-protien from associating
* BA targets GPCR for endocytosis
* receptor downregulation
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JAK Mechanism of Signaling
* regulatory molecule binds and brings two receptor molecules together to form a dimer
* phosphorylate each other
* activates JAK, allowing it to phosphorylate the receptor
* phosphotyrosine residues on the receptor proteins are binding sites for STAT proteins
* Signal Transducer and Activator of Transcription
* considered latent transcription factors
* phosphorylated by JAK
* Dimerize
* binds to specific sequences in the DNA
* Inactivation occurs when phosphatases remove phosphate groups from various proteins in the signaling pathway
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Molecules that work through JAK-STAT Signaling
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Drugs targeting the JAK-STAT pathway
* turn down the immune response
* cytokine receptor blocker derived from a monoclonal antiis used to prevent transplant rejection
* * binds to the IL-2 receptor.
* cytokine receptor blocker derived from a monoclonal antibody
* ruxolitinib
* inhibit the kinase activity of JAK
* treat a myeoloproliferative disorder
* Tofacitinib
* JAK inhibitor
* treatment of rheumatoid arthritis
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Examples of receptor tyrosine kinases
* molecules stimulating proliferation are growth factors
* many growth factors signal through receptor tyrosine kinases
* Examples:
* **Vascular-endothelial growth factor** (VEGF)
* promotes new blood vessel growth
* maintenance of endothelial cells in filtration membrane of kidney
* **Neurotrophins**
* promote the survival and differentiation of neurons.
* **Insulin-like growth factor-1 (IGF-1)**
* produced in response to growth hormone
* responsible for the majority of its growth-promoting effects.
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Receptor Tyrosine Kinase Steps in Signaling
* ligand binding causes receptor dimerization
* receptor autophosphorylation: receptors phosphorylate each other
* phosphotyrosine binding site for adaptor protein
* adaptor protein bound to a guanine-nucleotide exchange factor (GEF protein)
* GEF activates Ras, small monomeric G-binding protein
* GTP replaces GDP
* cascade of protein kinases
* phosphorylation of transcripition factors
* Ras control cell proliferation
* other monomeric G-proteins involved in
* Cell shape through effects on cytoskeleton
* involved in membrane trafficking through exocytosis and endocytosis
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Ras Inactivation
* GTP is hydrolyzed to GDP
* monomeric G-proteins like Ras are GTPases
* GTPase activity of Ras increased by GTPase activating protein or **GAP protein**
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Oncogene
* Mutant genes for proteins in these growth regulatory pathways are called **oncogenes**
* normal cells to cancer cells
* **proto-oncogene** is used to refer to the normal, unmutated form of the gene
* activating mutations of Ras in one third of tumors
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Nuclear Receptors
* ligand-activated transcription factors that bind nonpolar regulatory molecules
*
123
Nuclear receptor ligand examples
* steroid hormones
* thyroid hormones
* retinoic acid
* vitamin D3
* fatty acids
* phospholipids.
124
Orphan Receptors
physiological function and endogenous natural ligand are not known
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PPAR receptors
* involved in metabolic regulation
* Fibrates (which bind to the receptor PPAR-alpha) are a class of drugs used to treat dyslipidemia in order to lower the risk of cardiovascular disease
* Thiazolidinediones (TZDs; which bind to the receptor PPAR-gamma) are used to promote insulin sensitivity in the treatment of type 2 diabetes mellitus
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molecular structure of a nuclear receptor
* Three important domains
* transcriptional regulation domain
* DNA binding domain
* ligand binding domain.
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Signaling Mechanism Nuclear Receptors
* No ligand, inhibitory molecule binds the ligand binding domain
* Ligand bind--\> conformational change
* inhibitory molecule disscociates
* Nuclear receptor binds DNA
* Corregulator associates
* group protiens that regulate gene transcription
* Genes that are regulated by nuclear receptors contain particular DNA sequences (**response elements**) in their promoters, where the nuclear receptor binds
*
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Receptor Modulators
* One particular receptor may associate with different groups of coregulators in different cell types
* either enhance or inhibit transcription
* differential recruitment underlies the action of drugs known as **receptor modulators**
* Ex: **selective estrogen receptor modulator (SERM)** known as **raloxifene**
* estrogen receptor agonist in bone tissue, but estrogen receptor antagonist in breast and uterine tissue.
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NO
* uses
* how it synthesized/signals
* Neurotransmitter
* Paracrine
* relax smooth muscles in blood vessels
* Synthesis:
* nitric oxide synthase(NOS) is activated
* enzyme acts on an amino acid (L-arginine) to create nitric oxide
* Three forms NOS
* phagocytes, neurons, and endothelial cells of blood vessels
* NO diffuses to and activates a guanylyl cyclase
* soluable, cytosolic GC
* Guanylyl cyclase converts GTP to cyclic GMP
* cGMP acts as a second messanger
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Drug Actions Surrounding NO
* **Nitroglycerin** is converted to nitric oxide in the blood, which is why it dilates of blood vessels serving the heart.
* **Sildenafil **(Viagra®) block a form of phosphodiestase found in the penis
* cGMP not broken down
* more cGMP means more blood
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peripheral nervous system controls two types of effector organs
* skeletal muscles, which move the bones
* somatic
* and the viscera, which are all other organs
* autonomic
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autonomic nervous system organization
* The cell body of the **preganglionic neuron** is in the central nervous system
* the cell body of the **postganglionic neuron** ia found outside the central nervous system in an **autonomic ganglion**
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Parasympathetic vs. Sympathetic: Pre-ganglionic neurons
* parasympathetic
* preganglionic neurons in this case are in the brainstem or in the sacral spinal cord
* long, projecting to autonomic ganglia close to the visceral organ
* sympathetic:
* preganglionic neurons are in the thoracic and lumbar spinal cord
* axons tend to be short, with most autonomic ganglia lying close to the spinal cord
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Neurotransmitters and Receptors
* Para/Symp Pre and Post ganglion
* Neurotransmitter release
* Type of receptor activated
135
Ex: Autonomic Control of Heart Rate (Sympathetic)
* Speed heart rate
* preganglionic neurons are found in the **lateral horns** of the **first five thoracic segments** (approximately)
* axons of these neurons then project to the **sympathetic ganglion chain** (mainly in this case the **inferior cervical ganglion**) lying just outside the vertebral column
* in the ganglion the preganglionic neurons form synapses with the **postganglionic neurons**
* axons of the postganglionic neurons then project out of the ganglion and approach the heart along the large blood vessels connected to the heart
* spread over the surface of the heart before penetrating into the heart muscle tissue.
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Ex: Autonomic Control of Heart Rate (Parasympathetic)
* Slows heart rate down
* the cell bodies of the **preganglionic neurons** are in the **brainstem** (medulla)
* axons project out via the **vagus nerve**
* **next to the heart,** on the superior surface, are the cell bodies of the **postganglionic neurons**
* postganglionic axons are short, and project a short distance to the special cardiac muscle fibers near the top of the heart that control the heart rate.
*
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Ex: Autonomic Control of Pupil (Sympathetic)
* Dialates
* cell bodies of the sympathetic p**reganglionic neurons** again are in the **lateral horns** of the **upper thoracic segments**, but project mainly to the **superior cervical ganglion** of the sympathetic chain ganglia.
* In the ganglion are the cell bodies of the **postganglionic neurons**, and their axons then project up to the head and then through the superior orbital fissure and into the eye.
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Ex: Autonomic Control of Pupil (Parasympathetic)
* Constricts
* cell bodies of the parasympathetic **preganglionic neurons** are in the **brainstem** (midbrain) and their axons project out in the oculomotor nerve, which passes through the superior orbital fissure.
* On the other side of the fissure is the **ciliary ganglion**, which contains the cell bodies of the **postganglionic** neurons. Axons from these neurons then proceed to the iris.
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Where in general is the cell body of a preganglionic sympathetic neuron?
lateral horn
140
Where in general is the cell body of a postganglionic sympathetic neuron?
sympathetic ganglion
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Where specifically is the cell body of a postganglionic sympathetic neuron controlling pupil diameter?
sympathetic chain ganglion
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Where specifically is the cell body of a preganglionic parasympathetic neuron controlling pupil diameter?
brainstem
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Allergic rhinoconjunctivitis
* inhaled antigen binds to IgE bound to mast cells
* release of inflammatory paracrines
* histamine, certain arachidonic acid derivatives, and cytokines
* Immediately
* blood flow increases
* vessels become more permeable
* glands begins secreting fluid
* Several hours later
* influx into the tissues of neutrophils
* eosinophils
* basophils
* lymphocytes.
* Treatments include:
* oral antihistamines
* intranasal corticosteriods
* leukotriene inhibitors
* if symptoms are severe, oral corticosteroids.
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Atopy
excessive and abnormal IgE response
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acute urticaria
* when ingested antigens move through the blood and lodge in the skin
* producing red, itchy swellings called “hives”
* respond to antihistamines
* Genuine food allergies are also atopic
* Urticaria is one possible result
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anaphylactic shock
* Severe food allergies
* systemic release of histamine
147
eczema (atopic dermatitis)
* does not respond to antihistamines
* treated with glucocorticoids
* typical patient is a child with a tendency towards atopy
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Contact dermatitis
* Not atopic
* In response to chemicals from the environment, such as cosmetics, drugs or organic solvents
* Small molecules promote a cellular immune response against the skin after the small molecules bind to surface proteins on epidermal cells
* cellular immune response is slower than a one involving IgE
* rashes occur hours to days following the contact of the skin with the sensitizing agent
* delayed
* hay fever and other atopic allergies occur quickly
* need an initial sensitizing exposure
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Why not attack self?
* immature lymphocytes are deleted if they encounter antigen early
* clonal selection
* become tolerant to their antigen if they are steadily exposed to high concentrations
* ex: self antigens
* requires at least some stimulation of the innate immune response
150
Autoimmune disorders of based on T cell and antibodies
* SLE
* antibodies
* Type I DM
* T Cells
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myasthenia gravis
* antibodies
* bind to and inactivate the acetylcholine receptors on skeletal muscle
* The complexes are taken in by endocytosis and degraded, thereby reducing the total number of receptors on the muscle
* weak and fatigued muscle
* The complexes are taken in by endocytosis and degraded, thereby reducing the total number of receptors on the muscle
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Graves disease
* B-cells secrete antibody that binds to a protein
* stimulates the TSH receptor
* excess secretion of thyroid hormone
* goiter
* exophthalmos
* weight loss and various other problems associated with high utilization of energy
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Rheumatic Fever
* antibodies produced against a pathogen, in this case streptococci, cross-react with molecules in the body.
* Damage to the heart muscle and valves
* antibodies that are formed in response to molecules in the bacterial cell wall.
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Systemic lupus erythematosus (SLE)
* antibodies against a number of molecules, especially proteins associated with double stranded DNA
* Complexes of the antibody and antigen form and cause damage as they are deposited in tissues
* glomerulus of the kidney
* attacks multiple organs
* joints
* connective tissue structures such as the skin
* skin to sun readily damages the affected cells
* erythematous rash
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multiple sclerosis
* immune system responds to proteins in the myelin sheath of neurons
* Scattered islands (plaques) of demyelination occur in the central nervous system
* eventually replaced by plaques of hardened tissues, which can be observed through magnetic resonance imaging (MRI)
* "sclerosis" refers to "hardening"
* At first, symptoms are often mild
* relapsing-remiting-disorder
* progressive phase, in which there is increasing neuronal loss
* 3% chance each year of converting to the progressive phase
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rheumatoid arthritis
* immune system attacks the synovial membrane
* synovial inflammation and destruction of the structure of joints
* Both antibodies and T cells appear to be involved
* joint becomes inflamed, it undergoes hyperplasia and throws up finger like extensions
* extensive angiogenesis
* growing, abnormal, wound repair structure is called a pannus
* destruction of the adjacent bone and cartilage
* pannus entirely across the destroyed joint leads to ankylosis
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Ankylosing spondylitis
* prominent effects in the vertebral column
* “Spondylitis” refers to inflammation of the vertebra
* Ankylosis” refers to stiffening or fixation of a joint due to a disease process
* pain and stiffness in the spine and buttocks
* bony protuberances from the bodies of the vertebrae, and eventually this leads to complete fusion of the vertebrae
* Certain variations of HLA antigens (MHC molecules) are very common in many patients
* NSAIDs, immunosuppresives methotrexate and glucocorticoids, and blockers of TNF-alpha
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tetrodotoxin (TTX)
* blocks voltage-gated Na+ channel
* Japanese puffer fish, Fugu
* binds with high affinity and specificity to block the pore of the voltage gated Na+ channel found in the neurons and muscle cells
*
159
Local anesthetics
* lidocaine
* blocking the opening of the voltage-gated Na+ channel
* lower affinity for the channel
* much higher concentration is needed to effectively block the channel
* block nerve impulses along the axons of afferent neurons carrying pain information
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Cystic fibrosis
* mutations in the protein CFTR
* ABC protein
* Cl- (chloride) channel
* protein plays a key role in epithelial secretion
* transport of Na+ and Cl- into the lumen; water then follows by osmosis
* Flow of Cl- across the apical membrane through the CFTR channel is the rate-limiting and regulated step in this process
* mucus in the airways does not get sufficiently hydrated
* difficult to clear and causes clogs in the airways and degeneration
* environment that is conducive to the development of serious infections
* Treat with antibiotics
* autosomal recessive genetic disorder
* protein does not get transported from the rough endoplasmic reticulum (rER) to the apical plasma membrane
* digested via mechanisms in the rER designed to remove defective protein
* Drugs: CFTR correctors
* correct for this trafficking defect and restore expression of the protein on the apical plasma membrane
* Another type of mutation results in mutant proteins that are expressed on the cell surface, but that function poorly
* Ivacaftor (trade name Kalydeco)
* CFTR potentiator, that is, it prolongs the amount of time that the channel stays open
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adrenergic receptor
* important type of G-protein coupled receptors
* endogenous agonists (active chemical in the body that binds the receptor) are the catecholamines, norepinephrine and epinephrine
* Norepinephrine is the neurotransmitter released by sympathetic postganglionic neurons at their targets
162
two groups of adrenergic receptors
* alpha and beta
* alpha-1 receptors causing constriction of blood vessels
* beta-1 receptors in the heart that stimulate increased heart rate and force of contraction,
* beta 2 receptors that cause dilation of the airways
163
Affinity
* refers to the tightness with which a ligand binds to a receptor
* dissociation constant, Kd
* low Kd is indicative of a high affinity
164
efficacy
* refers to the range of effects of ligand binding on receptor activity
* and thus, biological response
* partial agonist
* do not produce the maximal biological response
* antagonists
* drugs that bind the receptor and prevent activation
* inverse agonists
* Drugs that inhibit basal activity
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Nicotinic acetylcholine receptors: Cholingeric Receptor
* ligand-gated ion channels
* rapid depolarization of the post-synaptic cell in response to acetylcholine
* postganglionic neurons at the synapses in autonomic ganglia and on skeletal muscle cells in the neuromuscular junction.
* Myasthenia gravis
* muscle weakness because the somatic efferent neuron has difficulty activating skeletal muscle fibers
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Muscarinic acetylcholine receptors
* G-protein coupled receptors
* act over a longer time course because more steps are involved before ion channels are opened or closed
* slow synaptic transmission
* found on the targets of parasympathetic postganglionic neurons, for instance the smooth muscle fibers of the circular muscles in the iris
* muscarinic antagonist to the eye in order to paralyze the circular muscles and prevent constriction of the pupil
167
Growth hormone (GH)
* stimulated by growth hormone releasing hormone (GHRH)
* peptide hormone that binds to a GPCR on GH-releasing cells in the anterior pituitary
168
acromegaly
* by excess GH secretion (hypersecretion)
* usually due to a tumor in the anterior pituitary
* contain a mutation in the G-alpha subunit
* lacks GTPase activity
* levels of cAMP
* stimulates GH hypersecretion
169
cholera
* chemically modifies the alpha subunit of the G-protein
* block its GTPase activity
* G-alpha remains active
* continually stimulating adenylyl cyclase
* to produce increased cAMP
* stimulates protein kinase A, which phosphorylates CFTR, the chloride channel involved in intestinal secretion
* greatly increases flow of Cl- through CFTR
* increasing intestinal secretion
170
Severe combined immunodeficiency (SCID)
* failure in the development of the immune system
* various genetic mutations
* X-linked gene coding for a subunit of the IL-2 receptor
* required for the development of B and T cells
* mutation in the gene coding for a JAK protein (JAK3
* treated with a bone marrow transplant
* bubble boy
171
neurofibromatosis
* mutation in a GAP protein
* prolonged Ras activity leading to the development of **neurofibromas**
* benign tumors that are derived from **Schwann cells**
*
172
HER2-positive breast cancer
* 18-20% of breast cancers are caused by overexpression of the receptor tyrosine kinase HER2
* Treatments:
* Monoclonal antibody drugs that bind to receptors
* Small molecules that are tyrosine kinase inhibitors
* trastuzumab (marketed as Herceptin)
* monoclonal antibody that binds to the extracellular domain of the receptor
* preventing receptor dimerization
* promoting receptor down-regulation
* Lapatinib (trade name: Tykerb)
* blocks growth factor signaling by preventing receptor phosphorylation
* targets epidermal growth factor receptors
Conj 401
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