wednesday rds,mTOR,cytokine, IF Flashcards
help understand rheumatology research rounds
What does mTORC2 do?
mTORC2 is an important regulator of cytoskeleton, F-actin stress fibers, PKC and PKB. MTORC2 binds MTORC1, MAPKAP1. PDPK1 phosphorylates PKB at serine 308 then MTORC2 phosphorylates PKB at serine 450, 473. PDPK-1 then phosphorylates PKB at S378 resulting in full PKB activation. Fully activated PKB can activate CREB, PI3K, and MTORC1. PKB inhibits p27, an inhibitor of cell cycle progression thus further stimulating growth. CREB (cAMP response element-binding protein) DEPTOR (DEP containing MTOR-inteacting protein) LST8, G protein beta subunit like, GbetaL, Gable MAPKAP1 mitogen activated protein kinase associated protein 1 p27 (CDKN1B gene product, cyclin dependent kinase inhibitor) PDPK1 (Phosphatidylinositol dependent kinase-1) can also phosphorylate other serine/threonine protein kinases SGK and atypical protein kinase C (aPKC, PRKCI gene). PI3K (phosphotidylinisotol 3 kinase) PKA (protein kinase A, cAMP dependent protein kinase) PKB (Protein kinase B, AKT, Akt-V-AKT murine thymoma viral oncogene homolog 3) PKC (protein kinase C) PROTOR protein observed with MTORC2 RICTOR rapamycin-insensitive companion of mammalian target of rapamycin SGK (serum/glucocorticoid -regulated kinase, first member of large subgroup of serine/threonine kinases). TEL2 telomere maintenance 2, homolog of TTI1 TTI1 TELO2-interacting protein 1
What is the difference between cytochrome C oxidase-complex 4, cytochrome b-C1, and cytochrome c?
Cyotchrome C oxidase is complex IV, a large integral mitochondrial membrane complex composed of 28 cytochrome subunits (COX-1 through 8) containing two hemes (cytochrome a and a3) and two copper centers using 14 assembly subunits. Cytochrome b-C1 is Complex 3 and contains 11 subunits, 3 respiratory subunits, 2 core proteins, and 6 low molecular weight proteins. Reduced ubiquinol donates electrons to ferricytochrome c which passes them to complex 4. Cytochrome C is a small protein, 100 aa, 17k daltons, with 1 heme unit that uses its Fe to shuttles electrons from Complex 3 to Complex 4. COX can mean cytochrome oxidase or cyclo-ooxygenase, renamed PTGS (ProsTaGlandin Synthase).
how does NF-kB work?
NF-kB refers to a cytoplasmic p50-RELA dimer generated from class I and II NFkB proteins. Both have N terminal DNA binding domains, class I has C terminal repressor activity, class II have activator domains. The DNA binding domains are bound by ikBa. NF-kB moves to nucleus when IkBa is phosphorylated and released. The preformed NF-kB transcription factor then binds response elements on multiple genes where additional factors mediate rapid transcription. p50-RELA dimer is the main TF combination. NFkB activates transcription of exogenous retroviruses. IkBa (Inhibitor of kappa B) NFkB (nuclear factor activated-kappa-light-chain enhancer of activated B cells) p50 generated by constitutive processing of p105, product of NFKB1 gene. relA product of RELA gene (V-Rel avian reticuloendotheliosis viral oncogene homolog A)
What is the peroxisome?
Peroxisomes are single membrane organelles drived from the endoplasmic reticulum that replicate by fission. A target signal on a protein determines content. Peroxisomes are involved in long chain fatty acids catabolism, biosynthesis of plasmalogens (ether phospholipids), and 10% of pentose phosphate shunt enzyme activity. Peroxisome contain oxidative enzymes-catalase, D amino acid oxidase, uric acid oxidase producing H2O2 (absent human). Peroxisomes oxidize about 25% of ingested ethanol.
What different types of blotting procedures are used to identify cellular molecules?
Southern blot uses known DNA and probes with DNA samples. A northern blot uses electrophoresis to separate cellular RNA, blots it onto nylon membrane, uses labeled RNA or DNA or oligonucleotide probes with at least 25 complementary bases to identify type of cellular RNA. A reverse northern plot uses isolated known DNA fragments on a membrane and probes with cellular RNA to develop an expression profile for that tissue. A Western blot separates unknown protein by electrophoresis on nitrocellulose and probes with a known antibody. Far-Western blotting probes a Western blot w post translational modification with a non-antibody binding protein to detect protein-protein interaction. An Eastern blot probes proteins separated by electrophoresis (SDS-PAGE gel) for post-translational modification to detect lipids, carbohydrates, or phosphorylation. A Far-Eastern blot (developed in Japan) uses (high-performance thin layer chromatography (HPTLC) to separate phospholipids which are then transferred to a membrane and probed by ligand binding or mass spectroscopy. Southern blot named after Edwin Southern.
What enzyme does metformin activate to inhibit mTOR?
Metformin inhibits mTOR by activating AMPK which decreases glucose utilization. Activated AMPK inhibits MTOR resulting in decreased fatty acid and protein synthesis, mitochondrial respiration, and up regulation of glycolysis, and autophagy- decrease anabolism, increase catabolism. AMPK (adenosine monophosphate-activated protein kinase)
What is the result of ROS generation by NADPH Oxidase?
ROS kills bacteria and fungi by inactivating critical metabolic enzymes, initiating lipid peroxidation, or liberating redox-reactive iron which then produces indiscriminate oxidants. SOD destroys ROS and inhibiting SOD increases ROS effectiveness. Knockout mice are more sensitive to lethal effects of superoxide inducing drugs. SOD (Superoxide dismutase)
What metabolic defect in substrate utilization can affect the electron transport chain to increase reactive oxygen species production?
Any defect that decreases the concentration of mitochondrial matrix NADH will result in excess of direct oxidation at complex 1, and 3 with increased production of reactive oxygen species.
How is tryptophan (C11H12N2O2) related to NAD?
Tryptophan is metabolized to kynurenine (C10H12N2O3) by tryptophan dioxygenase, an important step on the way to the synthesis of NAD (nicotinamide adenine dinucleotide-the nicotinic acid part).
What is the role of kynurenine in the immune response?
Kynurenine (C10H12N203) is a metabolic product of tryptophan. Dependent biological functions include dilating blood vessels during inflammation, and regulating the immune response. Synthetic L-Kyn in coculture of bone marrow-derived dendritic cells skews the differentiation of T cells to Treg cells rather than Th17 cells. Formation depletes tryptophan stores which depress growth of T cells and bacteria.
What is the difference between phospholipase C and protein kinase C?
Phospholipase C releases IP3 from PIP2 which then stimulates IP3 sensitive calcium channels on the ER. Protein kinase C is a serine threonine kinase that is activated by DAG and incased Ca++ (downstream from GPCR). It phosphorylates substrates near the cell surface and is important in signal transduction by phosphorylating ion channels.
What factors bind to Ras and alter its GTPase activity?
The Ras GTPase is the Ga subunit of the heterotrimeric G protein large GTPase found in G protein receptors. GTPase activating proteins (GAPs) encourage GTP to GDP transition. Guanine nucleotide exchange factors (GEFs) release GDP which is then replaced by the abundant cytosolic GTP. Son of sevenless (Sos) and cdc25 have a RasGEF domain. Ras activity depends on GTP: GDP ratio as well as factors like PI3K.
How do members of the Ras superfamily of proteins operate, and what do they do?
The Ras superfamily is a group of small GTPases related to the Ras subfamily (RAt Sarcoma source). Ras contains a GDP/GTP binding domain, and motifs for binding Mg, and guanine. The GTP form changes thnding affinity to other proteins that often increases that protein’s enzymatic activity. Receptor tyrosine kinases are a form of enzyme-linked receptors that activate Ras once phosphorylated by ligand binding. Members include: Ras controls cell proliferation, Rho controls cytoskeletal dynamics/morphology, Rab controls membrane trafficking, Rap controls cellular adhesion, Arf controls vesicular transport (ADP ribosylation factor), Ran controls nuclear transport, Rag RRAGA gene,Adenosine diphosphate ribosylation factor-1 GTPase ( member of the Arf small GTPase binding proteins of the Ras superfamily) is required for mTOR activation and lysosome localization by glutamine. Rheb activates mTORC1 kinase, and displays guanine nucleotide exchange activity. Miro controls mitochondrial transport.
What happens to the heterotrimeric G protein of the G protein coupled receptor?
Ligand binding separates the heterotrimeric G protein into 2 pieces, G alpha, the G beta bound to G gamma. Down stream enzymes are activated or suppressed, ion channels may also be affected. The alpha subunit ~354 aa in the resting state binds GDP. There are 4 families of G alpha subunits based on sequence homology. Gi family inhibit adenylate cyclase or activate phosphodiesterase. Gs family activate adenylate cyclase, Gq family activate phospholipase C, and the G12/13 family activate the Rho family of GTPases. The G beta gamma complex can attach to ligand gated ion channels (G protein-coupled inward rectifying potassium channels) or L-type calcium channels, or bind to histamine receptors and activate phospholipase A2 (release arachadonic acid from c2 of glycerol).
What are some downstream effects when AMPKK (AMPK kinase) activates AMPK? Not cAMP activated protein kinase (protein kinase A, PKA)
AMPK activation increases catoblism to create more ATP, and slows down anabolism to reduce ATP consumption. AMPK is activated when phosphorylated at threonine 172 by AMPKK, a complex of three proteins. Downstream effects include adipokines (Adiponectin and leptin) regulate appetite, metabolism, fatty acid catabolism, coagulation, and systemic inflammation may work through AMPK. Muscle cells upregulate oxidative metabolism via GLUT4, hexokinase 2, PPARalpha, PGC-1, UCP3, cytochrome C, and TFAM. AMPK (5’adenosine monophosphate-activated protein kinase) AMPKK AMPK kinase LKB1 a serine/threonine kinase MO 25 most protein 25 PGC-1 (peroxisone proliferator-activated receptor gamma co-activator 1-alpha) PPARalpha (peroxisone proliferator-activated receptor alpha) TFAM (mitochondrial transcription factor A) UCP3 (mitochondrial uncoupling protein 3)
What are the phospholipases A-C and the phosphokinases A-C?
Phospholipase A1 catalyzes the cleavage of the sn-1 position of phospholipids, forming a fatty acid and a lysphospholipid. Phospholipase A2 cleaves the SN-2 position (release arachadonic acid). Phospholipase B can cleave both the SN-1 and SN-2 positions.Phospholipase C catalyzes the cleavage of I3P. Phosphokinase A does not exist, phosphokinase B is aKT and phosphokinase C is a serine/threonine kinase activated by binding to diacylglycerol.
How does mTORC1 increase production of reactive oxygen species?
mTORC1 increases cytosolic glycolysis and decreases oxidative phosphorylation thus lowering O2 demand and allowing excess O2 to form reactive oxygen species.
What is the difference in function between mTORC1 and mTORC2?
MTOR forms different complexes due to multiple protein binding domains (eg PH, TPR domains) that affects substrate access. mTORC1 acts as the nutrient/energy/redox sensor which is stimulated by insulin, growth factors, serum, amino acids, and oxidative stress. Phosphorylates S6 kinase 1 - S6 ribosomal protein. mTORC2 is an important regulator of cytoskeleton, F-actin stress fibers, PKC and PKB phosphorylation. MTOR -/- knockouts die as embryos, -/+ are normal and fertile. PKC (protein kinase C) PKB (protein kinase B, Akt) MTORC1 expands IL-4 double negative T cells, Th-17 cells, and contracts Tregs in SLE. PH domain- plextrin homology domain. TPR (tetratricopeptide repeat) ~34 aa seq;uence In SLE, rapamycin inhibits mTORC 1 in Tregs and promotes expansion of Tregs. -Kato
How do tuberous sclerosis proteins affect mTOR?
TSC2 is a peripheral membrane protein that combines with TSC1 in cells to become a cytosolic dimer with GTPase activating protein (GAP) activity that inhibits rheb GTPase by dephosphorylatiion. TSC2 unbound by TSC1 activates Rheb (GTPase) which then activates mTOR by removing the inhibitory protein FKBP8 which binds FRB motif and prevents kinase activity. Otherwise amino acids can stimulate mTOR directly by Rag C/D binds GTP ->Rag A/B, binds to lysosome, attracts Rheb which activates mTORC1. The TSC1/TSC2 heterodimer is a sensitive to upstream signals (AKT) that result in increased or decreased mTOR activation and can function as a tumor suppression gene when dimerized. TSC2 (tiberpis sclerosis complex 2, tuberin, tuberous sclerosis complex refers to disease not a complex of proteins) TSC1 (hamartin, peripheral membrane protein) rheb (RAS homolog enriched in brain), membrane bound farsenylated FKBP8 (FK406-binding protein 8, FKBP8, 38KD, FKBP38)
How does mTORC1 inhibit autophagy?
Activated mTORC1 phosphorylates Atg 13 which prevents autophagosome construction at the plasma membrane. Atg 13 (autophagy related protein 13)
What does MLST8 (MTOR subunit LST8) do in the MTORC1 complex?
MLST8 binds to kinase domain of MTOR and stabilizes interaction with RPTOR (raptor). Necessary for phosphorylation of S6K1 and 4EBP1. MLST8 (mammalian lethal with SEC protein lethal 8) RPTOR (regulatory-associated protein of MTOR
how are various domains distributed by location along the MTOR molecule?
The MTOR complex binds multiple proteins. N terminal end has multiple HEAT repeats comprised of TPR motifs, then one FAT domain, then FRB domain then more TPR repeats. A class IV PI3K/PI4K domain is near the C terminal end, then a cFAT domain. MTOR consists of 2,549 aa, and calculated mol wt of 300 kD. ATM (ataxia telangiectasia mutated) cFAT (C terminal FAT domain) FRB (FKBP12-rapamycin binding domain). FAT [FRAP-ATM-TRRAP) FRAP-(FKBP-rapamycin-associated protein) HEAT (Humtingtin, Elongation factor 3 (EF3), protein phosphatase 2A (PP2A), ATM, yeast kinase TOR1) PI3K/PI4K class IV phosphatidylinositide-3-kinase, serine/threonine kinase MTOR (Mechanistim Target of Rapamycin; MTOR Mammalian Target of Rapamycin, FRAP (FKBP12-binding protein, 12-rapamycin complex-associated protein 1), RAFT1) TPR (tetratricopeptide repeat) ~34 aa seq;uence TTRAP (transformation/transcription domain-associated protein member of the phosphatidylinositol 3-kinase-related kinase protein family)] domain.
How does MTORC1 complex work?
MTORC1 works by having bound proteins that restrict serine/threonine enzymatic activity to proteins that stimulate cell proliferation and increase cell size only when metabolically appropriate. MTORC1 is 1Md in size, contains 2 hetero tetramers with rhomboid shape and central cavity formed by 2 MTOR and 2 RPTOR, MLST8 subunits, which form distal foot like processes. AKT1S1 is in central core and has 10 serine/threonine sites where phosphorylation by PKB, regulates kinase activity by causing AKT1S1 to leave the complex and permit substrate access to MTORC1. Also binds ATP, RNA polymerase, and attaches to mitochondria, endoplasmic reticulum, golgi. lysosomes, endosomes, pml body in nucleus. The FRB motif on MTOR binds FKBP1A (FKBP12), forming catalytic cleft in MTORC1. The FKBP1A-rapamycin complex does not form an enzymatic site. FKBP8 (FKBP38) is related to FKBP12 and inhibits MTORC1 similar to FKBP 12-rapamycin. Rheb contains 5 GTP binding repeats,- has GTPase activity, and once activated displaces FKBP8 and thus activates MTORC1. AKT1S1 (AKT1 Substrate 1, Proline rich, PRAS40) MLST8 (MTOR subunit LST8) MTOR ( mechanistic target of rapamycin, FKBP12-rapamycin complex-associated protein 1, (FRAP1)) PML (promyelocytic leukemia bodies) PKB (AKT1) RPTOR (regulatory-associated protein of MTOR 1335 aa)
What is RPTOR?
RPTOR (regulatory-associated protein of MTOR) which regulates growth, survival, and autophagy. It serves as a scaffold for recruiting MTORC1 substrates, and various kinases. Binds 14-3-3 protein, protein kinase, RNA polymerase 3. Has 10 serines phosphorylated by MAPK8, RPS6KA1, MTOR. Insulin induced phosphorylation regulates MTORC1. Growth factors up regulate MTOR via RPS6KA1 by phosphorylating serines at 3 sites . Nutrient limitation results in RPTOR phosphorylation by AMPK which associates with 14-3-3 which down regulates MTOR. RPTOR binds directly to 4EBP1 and RPS6KB1, independent of MTORC1 . Interacts with SPAG5and G3BP1 which is increased by oxidative stress. Interacts with HTR6. Contains 7WD repeats 14-3-3 binds to over 200 signaling proteins MAPK8 mitogen activated protein kinase 8 RPS6KA1 ribosomal protein S6 kinase alpha 1 SPAG5 (Sperm associated antigen 5) G3BP1 (RasGTPase-activating protein-binding protein 1) HTR6 (5-HT (serotonin) receptor 6) WD repeats (about 40 amino acid motif culminating with tryptophan-aspartic acid-W-D) forms circular solenoid protein domain (WD-40 domain).
how do amino acids activate mTOR?
In the presence of amino acids Rag proteins become RagC GDP and RagB GTP, and attracts mTOR and Rptor to become mTORC1. P62 associates with RagC GTP and RagG GDP on the surface of a lysosome. P62 (Sequestosome 1; SQSTM1 gene) rags (GTPase-activating protein for Rag subunits A/B)
How do enzymatic complexes coordinate incoming signals prior to gene activation? What is the ragulator complex?
Enzymatic complexes coordinate different processes by binding different proteins according to different conditions resulting in different transcription factors. Conditions cause post transitional modifications -phosphorylation, GTP binding so that the complex localizes to different sites and attract different mix of activators and suppressors. The ragulator complex composed of MAPKSP1, ROBLD3 and c11orf59 interacts with Rag GTPasees to recruit mTOR to the lysosomal surface where it becomes active by binding Rheb. MAPKSP1 (Mitogen activated protein kinase scaffold protein 1) enhances signal. ROBLD3 (? p14, Lamtor2, endosomal?lysosomal adaptor MAPK and MTOR activator 2) c11orf59 (chromosome 11 open reading frame 59, late endosomal/lysosomal adapter, MAPK and MTOR activator 1)
How do gator complexes affect rag activity?
Rag activity is regulated by GATOR complexes. GATOR1 contains DEPDC5, Nprl2 and Nprl3. GATOR2 complex contains Mios, WDR24, WDR59, Seh1L, and Sec13. GATOR1 inhibits Rags. GATOR2 activates Rags by inhibiting DDPDC5. DEPDC5 (DEP domain-containing protein 5) GTPase activator GATOR (GTPase activating protein activity towards RAGs) Nprl2 gene TUSC4(tumor suppressor candidate 4, protein phosphorylation, GTPase activator. Nprl3 (nitrogen permease regulator-like 3) Mios (missing oocytte/meiosis regulator, Drosophila, homolog of) WDR24 (WD repeat containing protein 24) WDR59 (WD repeat containing protein 59) rags (GTPase-activating protein for Rag subunits A/B) Seh1l (Seh1-like protein) localizes nucleoporins to nuclear envelope Sec13 (sec13-like protein 1,sec13L1) suppresses mTORC1 signaling.
What is the role of GATA-3 in the Th2 response?
GATA-3 is a zinc finger transcription factor that promotes differentiation of Th2 cells and secretion of IL-4, -5, -8 and -13. Suppresses differentiation to Th1 cell line. Defects result in hypoparathyroidism with sensorineural deafness and renal dysplasia. Often expressed in breast cancer- luminal type. GATA-3 expression is increased in CD8 lupus T cells and is insensitive to rapamycin. IL-17 but not IL-4 induces suppression of GATA-3. GATA-3 (enhancer binding protein GATA-3, Trans acting T cell specific transcription factor)
IL-23 Is composed of what proteins and stimulates what other interleukin?
IL-23 is composed of IL-12p40 subunit shared with IL-12 and the IL-23p19 subunit. IL-23 simulates T helper cells producing IL-17 and delayed type hypersensitivity. IL-23 contributes to experimental autoimmune inflammation in numerous models of encephalitis, arthritis, enteritis, and psoriasis.
What is the result of IL-17 stimulation and which cells are most sensitive?
IL-17 induces production of cytokines IL-6, G-CSF, GM-CSF, IL-1 beta, TGF-beta, TNF alpha and chemokines IL-8, MCP-1, and prostaglandins PGE2 for many cell types including fibroblasts, endothelial cells, epithelial cells, keratinocytes, and macrophages. Stimulates Th17 cells linked to many autoimmune diseases. IL-17 expression is increased in CD4 positive T cells and is suppressed by rapamycin. Neutralization of IL 17 but not IL-4 expanded Tregs in SLE and HC subjects-Kato.
What is the fate of immature B cells once they leave the bone marrow ?
Circulating immature B cells are CD45, CD19, CD40, IgM BCR+ that has undergoes deletion or receptor edit, before leaving the bone marrow. B-1a lymphocytes are CD5+CD11b/CD18+CD45RAlow, and produce low affinity poly-reactive IgM Ig using germline genes. They are self replenishing, are found in the blood in peritoneal cavity. CD5 ligation induces TNF and IL-2R. B-1b have inducible CD5, are CD11b/CD18 +, CD45RA low, produce high affinity IgG, IgM, and IGE immunoglobulins, found in the bone marrow and blood, and CD5 ligation causes apoptosis. B-2 lymphocytes are conventional, have inducible CD5, CD11b/CD18 -, CD45RA high, and undergo further differentiation in lymph nodes and apleen. Fo B express IgM, IgD, CD21, CD19, and CD81. organize into primary B follicles, white pulp spleen, cortical areas lymph node. freely recirculate. CD21 binds C3 and with CD19 and CD81 produce co-receptor complex which increasea BCR responsiveness. Fo B-follicular B cell CD5 has an ITIM motif which reduces antigenic responsiveness,. No confirmed ligand, may be homophilic and bind to itself. CD11b/CD18 (integrin alpha 1 ITGAM MAC-1 CR3) / CD1b (Thymocyte antigen) CD18 (integren beta chain beta 2) CD19 transmembrane Ig like extracellular, intracellular tyrosines affect BCR responsiveness once phosphorylated. CD21 complement receptor CR2 CD81 transmembrane glycoprotein, signal transducer CD45RA (Protein tyrosine phosphatase receptor type C PTPRC)
What are the differences between B1, B2, and Fo B (follicular) lymphocytes?
B-1 cells have more IgM than IgG on the surface with low antigen avidity responding mostly to polysaccharides. Present in pleural and peritoneal cavities. B-2 cells are intended when B cell is otherwise unqualified. T follicular helper cells, help for primary immune response, isotype switching. no CD1 or CD5, different from B1 B cells, and marginal zone B cells.
What does interferon inducible GTPase do?
Interferon inducible GTPase is a family of proteins primarily associated with the golgi apparatus and endoplasmic reticulum which affects intracellular membrane trafficking. Immunity-related GTPase family M protein (IRGM) is a member which regulates autophagy in response to intracellular pathogens.
