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What is a leader sequence?
Sequence of 16-20 AAs in some eukaryotic cells at N-terminus - determines destination. None in proteins in cytosol.
What is the signal peptide?
leader sequence peptide
How do leader sequences function?
Leader sequence of protein destined for ER contain hydrophobic AAs that become embedded in lipid bilayer membrane - functions to guide incoming (nascent) protein to receptor protein that marks position of pore in membrane. Once protein passes into cisternal lumen through the pore - leader sequence is cleaved.
What adaptive cells do not need APCs?
B cells
What cell makes Abs?
Effector cells/Plasma cells - secrete Abs (immunoglobulins)
What do B cells do upon recognition of an Ag?
undergo activation, proliferation, and differentiation into plasma cells - plasma cells produce large amounts of Ab specific for pathogen
What do Abs do?
- neutralisation of the pathogen
– opsonisation
– complement fixation
DO NOT destroy pathogen - only mark it
How do T cells recognise epitopes (antigen fragments)?
antigen broken down into peptides (as epitope is hidden) - which binds to MHC molecule - T cell receptor binds to this complex of MHC and epitope peptide - CD4+ = extracellular; CD8+ = intracellular
How do B cells recognise epitopes (antigen fragments)?
amino acids or even sugar residues on surface of antigen
What does ITAMs stand for?
Immunoreceptor Tyrosine-based Activation Motifs
What are ITAMs?
signalling motifs found in cytoplasmic domain - Yxx[L/I]x(6-8)Yxx[L/I] (Y = tyrosine, L/I = leucine/isoleucine, x = AAs)
What is the ITAM containing receptor on T cells?
CD3ζ chains
What is the ITAM containing receptor on B cells?
CD79alpha/CD79beta
What is the ITAM containing receptor on Fc?
macrophages, NK cells, and mast cells
What are the B cell coreceptors?
CD19, CR2 (CD21) (recognises iC3b and C3d), CD81
What is the function of CD81
brings CD19 to the surface membrane
How are B cells activated?
CR1 on B cell binds to C3b bound on antigen - cleaved by factor I into C3d and iC3b; CR2 then binds C3d; Lyn (tyrosine kinase) phosphorylates CD19 cytoplasmic tail, binds intracellular molecules and sends activation signal
What are FDCs?
follicular dendritic cells (stromal, non-hematopoeitic) found in B cell follicles - essential for B cell maturation, non phagocytic, express CR2 and to a lesser extent CR1, bind to C3b which is cleaved by factor I to CD3
What are TI agents?
thymus independent agents: TI-1 and TI-2
What are TI-1?
additional signalling: TLR-4 binds to LPS, TLR-9 binds to bacterial DNA
What are TI-2?
repetitive carbs or protein epitopes at high level on pathogen surface; cross-links BCR and co-receptors extensively - no need for additional signals; e.g. cell wall polysaccharides of Streptococcus pneumoniae
What begins in the bone marrow?
except FDCs; B and T cells, mast cells, neutrophils,
What cells are located in the blood?
lymphocytes, monocytes, neutrophils, eosinophils, basophils
What cells are located in the lymph?
lymphocytes, DCs
What cells are located in organs/tissues?
DCs, macrophages, T and B cells (mast cells and lymphocytes
What are the primary lymphoid organs?
thymus (t cells), bone marrow (b cells)
What are the secondary lymphoid organs?
spleen, MALT, GALT, BALT, tonsils, lymph nodes,
What happens in inflammation?
tissue damage - mast cell degranulation - release of cytokines, histamine, prostaglandins and leukotrienes - vasodilation - increased blood volume (calor and rubor) - increased vascular permeability; fluid leakage (tumour) of complement, cytokines, Abs and clotting factors - adhesion of leukocytes (neutrophils, monocytes which differenciate to macrophages) to endothelial tissue of blood vessel wall - guided to site (chemotaxis) - release of chemokine signal induces this
What are anaphylatoxins (examples)
C3a, C4a, C5 - complement peptides; increase vascular permeability, immune cell recruitment, degranulation
What are the costimulatory molecules?
CD40L, CD40 (B cells and FDCs)
CD28 and B7 (T cells and DCs)
How does Ag processing work in MHC Class II molecules?
extracellular antigen - peptides produced in phagolysosome - MHC class II molecule produced in ER - binds to peptides in vesicle - transported and presented at surface membrane
How does Ag processing work in MHC Class I molecules?
intracellular organism - Ag processing in proteosome - transported to ER - binds with MHC Class I molecule - transported and presented at membrane
MHC Class I pathway?
cytosol - proteolysis through proteosome - TAP - surface membrane
MHC Class II pathway?
endosome/lysosome process Ag - MHC class II synthesised to form alpha/beta chains and li - chain releases li to bind exocytic vesicle to endosome - presented to surface
Ovalbumin & Glutaraldehyde Treatment?
T cells can still bind to pre-digested peptide fragments of ovalbumin then fixed with gluteraldehyde - instead of native (whole Ag)
CTL experiment?
better recognise short linear peptide sequences than envelope proteins (nucleoproteins (internal) v hemagglutinin)
Cells that display peptides in class II molecules?
APCs
Cells that display peptides in class I molecules?
target cells
How can cells be APCs?
constitutively express MHC Class II and costimulatory molecules to naive Th cells - must be activated by phagocytosis before producing MHC Class II and B7 - express MHC Class II but must be activated before presenting B7
What proteins experience continuous turnover and degradation at a rate expressed by their half-life?
Transcription factors
Cyclins
All key metabolic enzymes
Denature, misfolded and abnormal proteins
What identifies proteins for the proteosome?
ubiquitin (released by proteosome) - proteolysis occurs
What are the three steps of ubiquitin attachment?
E1 activation - E2 conjugation - E3 ligation
What are the cytokines released by T cells?
IL-2-3-4-5-10 and IFN-gamma
What is the structure of a proteosome?
20S catalytic core made of four stacks rings of 7 subunits - outer rings = alpha (structural); inner rings = beta 3-4-6-7 (structural)
beta 1, 2 and 5 = proteolytic subunits
caspase-, trypsin- and chymotrypsin- like
cleaves acidic, basic and hydrophobic
19S regulatory caps on either end
When complexed = 26S at sedimentation (centrifugation)
10-50 angstrom in diameter
Active sites of proteolytic proteins on outer rings
What does 19S do?
recognises ubiquitin and opens to allow into catalytic core which unfolds proteins
What happens in the proteosome?
Peptide bonds cleaved by AA sequence; ATP-dependent process
What do proteosomes replace B1, 2 and 5 with?
two MHC Class II molecules; LMP2, and LMP7 and one non-MHC Class II molecule LMP10
LMP2 (beta1i)
LMP7 (beta5i)
LMP10 (MECL-1, beta2i)
What induces this?
IFN-gamma (produced by NK cells)
What is the carboxy terminus anchor residue?
hydrophobic C terminus of peptide that allows binding to MHC Class I molecule
What does IFNgamma allow?
further modification for the binding of PA28 (proteosome activator complex)
What is PA28?
a 6 or 7 membered ring - two; alpha and beta - that bind to 20S core instead of 19S - increases release rate of peptides
Creates immunoproteosome
What is TAP?
membrane-spanning deterodimer
TAP1 and TAP2
transports AAs, sugars, ions and peptides
ATP-binding cassette proteins (ABC domain in cytosol)
hydrophobic transmembrane domain through ER
transports peptides (optimal for MHC Class I: 8-10 AAs) from immunoproteosome in cytosol to ER
What can TAP deficiency cause?
Bare Lymphocyte Syndrome Type I (BLS I)
Autoimmunity (overproduction of NK cells as compensation)
Immunodeficiency (no MHC Class I molecules - no CTL activation)
What aids MHC molecule binding?
Chaperone molecules e.g. calnexin (membrane protein of ER)
What does calnexin do?
associates with free alpha chain + promotes folding
when beta2 chain binds to alpha chain, calnexin released
chaperone molecules calreticulin and Erp57 (forms disulphide bond with tapasin and stabilises calreticulin) and tapasin bind
tapasin binds complex to Tap
released when stable and MHC complex transported from ER through golgi to surface membrane
What are DRiPs?
defective ribosomal products (degraded into peptides)
What is the PLC?
Peptide loading complex (calreticulin, Erp57, tapasin)
Once stable peptide is bound, all released
What does ERAP do?
Removes N terminal AAs from peptide bound in MHC Class I molecule to make length 8-10 AAs long (optimal)
What are the three intracellular membrane bound vesicles in endogenous endocytic pathway?
early endosomes (pH 6-6.5)
late endosomes (pH 5-6)
lysosomes (pH 4.5-5)
How are proteins degraded here?
encounter increasingly acidic environments;
hydrolytic enzymes degrade into oligopeptides of 13-18 AAs
How is Ag processing inhibited?
chemicals that increase pH as well as protease inhibitors
How does T cell activation relate to this pathway?
different APCs may degrade same native proteins into distinct peptides due to different endosomal proteases;
these are specific for activation of different T cells
What do invariant chains do?
Ii chain binds to MHC Class II molecule in RER, preventing any endogenous peptides from binding (only allows MHC Class II in endocytic pathway)
Slowly degraded through increasingly acidic endosomes, leaving CLIP (Class II associated invariant chain peptide) which remains until peptide bound preventing premature Ag binding
How do HLA-DM and HLA-DO function?
HLA-DM degrades CLIP (weakly bound peptide) to allow high affinity Ag peptides to bind to MHC Class II
HLA-DO reduces efficiency of HLA-DM in immature APCs and thymic epithelial cells (to prevent immature peptide binding)
HLA-DO is decreased during inflammation/infection
HLA-DO has an affinity for certain peptides
Examples of opsonins
C3b, C4b; CR1
IgG Abs, MBL; FcgammaR
CRP; FcgammaR and C1q
