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DNG THC L TRNH TN HIU (SIGNALING TRANSDUCTION CASCADES) C ch ca l trnh tnh hiu

Sau khi c s tm hiu chi tit v tng loi th th mt, by gi ta s cng t chng trong mi quan h tng ha ca mt s l trnh tn hiu (signaling transduction) di quan im ca ha sinh phn t (molecular biochemistry) c c ci nhn hon chnh t phn t n chc nng. L trnh tn hiu hiu mc t bo c ngha l s chuyn thng tin t bn ngoi vo trong t bo. S truyn tn hiu c th n gii nh cc qu trnh lin quan n th th ca acetylcholine (nhng th th c cu to knh, thng qua s tng tc vi ligand s cho php tn hiu i qua di dng mt ion nh vo hoc ra khi t bo. S di chuyn ca nhng ion ny lm thay i in th mng v lan ta trn ton th t bo ). L trnh tn hiu phc tp hn lin quan n hng lot cc p ng khc nhau k t khi ligand ni kt vi th th. Trong c th k n s phosphoryl ha ca tyrosine kinase hoc/v serine/threonine kinase. S phosphoryl ha protein thay i hot tnh ca enzyme v s hnh thnh ca cc protein c lin quan khc, do thay i hot ng ca t bo v qu trnh biu hin gene, gy ra s p ng t bo.

Phn t tn hiu
Phn t tn hiu l nhng phn t ha hc c tit ra t mt t bo c kh nng khi ng mt chui phn ng sinh hc ni bo qua trung gian th th. Do vy c th xem n nh mt phng tin lin lc ca t bo, c gi l SM (signaling molecule). Cc SM c to ra t bo kim sot hot ng ca chnh n (autocrine) hoc ln cn (paracrine) hay phng thch vo mu n kim sot hot ng ca t bo khc (endocrine). S khc bit c bn gia cc hnh thc ny l thi gian, paracrine v autocrine cho p ng tc th, cn endocrine cho p ng sinh hc chm hn nhiu. C th xem y nh mt ligand. C 2 c tnh c bn sau: Cc ligand c th gn kt vi nhiu loi th th khc nhau sinh ra cc p ng thuc cc loi khc nhau. V d: SM nh NE c th gn vi th th 1 thc hin phng thc cn tit hoc 2 thc hin phng thc t tit. Nhiu ligand c th gn kt vi cng mt loi th th sinh ra cc p ng ging nhau. V d ligand gn vo th th 1, ET (th th endothelin) v AT1 (th th ca angiotensin II) u hot ha PLC (Phospholipase C) qua protein Gq ri bin i PIP (phosphatidyl inositol biphosphate thnh phosphatidyl inositol triphosphate (PI3) mt mt gii phng Ca2+ li ni bo, mc khc hot ha PKC (phospho kinase C) phosphoryl ha knh calcium loi L lm m knh ny.

Ngoi ra cn mt hnh thc khc trong phn loi v s tng quan gia cc phn t tn hiu v c quan ch c lm r qua v d sau. d dy, gastrin c phng thch vo mu hot ha t

bo ECL (enterochromaphil-like cell) tit ra histamine, sau histamine kch thch bm proton thnh t bo phng thch ion H+, nh th s lin lc gia t bo G vi t bo ECL l endocrine trong khi ECL vi t bo thnh l paracrine.

Phn loi th th
Cc th th ca l trnh tn hiu (signal transducing receptor) c phn thnh 3 nhm c bn: Th th xuyn mng v c hot ng sinh enzyme ni bo (intrinsic enzymatic activity). Nhm ny gm c tyrosine kinase (th th PDGF, insulin, EGF v FGF), tyrosine phosphatase (CD45 ca t bo T v i thc bo), guanylate cylase (natriuretic peptide receptor) v serine/threonin kinase (th th activin v TGF-).Nhng th th tyrosine kinase ni bo c kh nng t phosphoryl ha cng nh l phosphoryl ha cc c cht khc. Ngoi ra cng c mt vi h th th thiu hot ng sinh enzyme ni bo nh cp (coupled) th th tyrosine kinase ni bo, chng c vai tr trong s tng tc proteinprotein. Th th c dng cp (coupled), trong t bo, kt ni vi GTP v thy phn protein (termed G-protein). Th th thuc nhm ny tng tc vi G-protein v tt c u c cu trc 7 vng xon xuyn mng. Nhng th th ny cn c gi l th th xon (serpentine receptor). Bao gm adrenegic, odorant v cc th th hormone (glucagon, angiotensin, vasopressin v bradykinin). Nhng th th c tm thy ni bo v ligand s kt ni vi th th ti b mt nhn, s tng tc s dn n qu trnh chuyn m gene. Bi v cc th th thuc nhm ny c tm thy ni bo v hot ng chc nng ngay trong nhn nn cn c gi l th th nhn (nuclear receptors). Nhng th th ny bao gm mt lng ln thnh vin thuc cc h th th hormone steroid v thyroid. Cc th th ny c vng ni kt ca ligand, vng ni kt DNA v vng kch hot chuyn m.

Do gii hn ca ti, y ch trnh by s iu ha bi s phosphoryl ha, cc tc ng xem xt mc phn t dng thc tn hiu (signal cascades) ca G-protein, cAMP v mt vi cht dn truyn th cp khc sinh ra t phosphatidylinositol.

7.1 Kinase v phosphatases:


C nhiu enzyme c iu ha bi lin kt cng ha tr vi gc phosphate, lin kt ester, lin kt vi gc hydroxyl ca mt vi amino acid (serine, threonine hoc tyrosine).

Protein kinase chuyn gc phosphate t phn t ATP n gc hydroxyl ca protein. Ngc li, protein phosphatase thy phn lin kt phosphate. Cc enzyme c iu ha bi PKA

S phosphoryl ha thay i trc tip hot tnh ca enzyme bng cch thay i cu trc cu to ca n. Ngoi ra, s thay i hot tnh cng xut pht t s ni kt mt protein vo v tr phosphate ha trc . V d, 14-3-3 protein ni kt vo vng c serine v threonine phosphate ha chui RXXX[pS/pT]XP, X c th l mt amino acid no . S lin kt vi 143-3 l c ch thng xy ra cc protein c vai tr nh mt yu t chuyn m (transcription factors) , chng thng c gi li trong bo tng v khng vo c trong nhn. Protein kinase v phosphatase c kh nng t iu ha thng qua mt chui truyn tin phc tp. V d: Mt vi protein kinase c kch hot bi h thng Ca2+ - calmodulin hay protein kinase A (PKA) c kch hot bi cAMP.

Nh ta bit, adenylate cyclase thy phn ATP to thnh cAMP v PPi. S ni kt hormone ngoi bo (v d nh epinephrine) kch hot adenylate cyclase to thnh cAMP trong bo tng. Do vy, cAMP c th gi l cht truyn tin th hai hay cht truyn tin th pht (second messenger).

Hnh 7.1: Minh ha c ch phn ng cAMP.

Hnh 7.2: Minh ha s khi c ch hot ng ca cAMP Phosphodiesterase thy phn cAMP thnh AMP do vy n khng th tip tc hot ha thy phn protein kinase A c na. Ngi ta xc nh c rng cAMP kch thch qu trnh t ging cp ca mnh (cAMP stimulates its own degradation) nhanh chng bt hot tn hiu ca chnh n. Protein kinase A (protein kinase ph thuc cAMP) chuyn gc Pi t ATP n gc hydroxyl ca nhm serine hoc threonine, y l phn c th ca trnh t 5-amino acid chuyn bit. Protein kinase A tn ti trng thi tnh (resting state) cu trc nh sau: 2 tiu n v iu ha (R). 2 tiu n v thy phn (C).

Hnh 7.3:Lt trnh byOTR illustrating putative potential phosphorylation sites. The putative phosphorylation targets are indicated as (1) calmodulin II kinase (CaM II K), (2) protein kinase C (PKC), (3) cAMP-dependent protein kinase A (PKA), (4) casein kinase Ia(CK-I) and (5) cGMP-dependent protein kinase G (PKG). Bnh thng, mi tiu n v iu ha ca protein kinase A c cha trnh t pseudosubstrate (c cht gi) c th so snh c c vi vng cht nn (substrate domain) ca protein ch ca protein kinase A, nhng trong cu trc ca n alanine thay th cho serine hay threonine. Vng pseudosubtrate ca tiu n v iu ha thiu gc hydroxyl do vy khng phosphate ha c, gn vo vng hot ng (active site) ca tiu n v thy phn v kha hot ng ca n. Khi mi tiu n v iu ha lin kt vi 2cAMP, s thay i cu trc ca vng ny s dn n vic gii phng tiu n v thy phn. Hai tiu n v ny sau c th phosphoryl ha thy phn ha serine hoc threonine ca protein ch. R2C2 + 4cAMP R2CAMP4 + 2C

Hnh 7.4: S lc s hot ha PKA Lu rng, PKIs (protein kinase inhibitors) iu ha hot ng ca tiu n v C.

Hnh 7.5: Bisindolylmaleimide mt protein kinase inhibitor. Cu trc tng t nh staurosporine, c tnh chn lc cao i vi protein kinase C (PKC) v l cht c ch cnh tranh.

Hnh 7.6: Minh ha c ch phn gii glycogen lm tng glucose mu.

7.2 Dng thc tn hiu ca G-protein


Hu ht cc phn t tn hiu (signal molecules) ni kt vi th th trn mng t bo, ch c cc phn t tn hiu thuc nhm steroid hoc mt s nhm km phn cc khc mi xuyn mng tng tc vi cc th th ni bo (intracellular receptors).

C mt h (family) th th b mt ln c motif chung 7 vng xon xuyn mng (7 transmembrane a-helices), cng xem 2 v d di y: Rhodopsin l th th u tin c tm ra bi k thut tia X tinh th hc (X-ray crystallography). Th th -adrenergic c kch hot bi epinephrine v norepinephrine. S kt tinh (crystallization) ca th th ny c cng c bi mt ni kt c hc (insertion) ca lysozyme (enzyme tan trong nc) vo quai bo tng (cytosolic loop) xen gia cc xon xuyn mng.

Hnh 7.7: Minh ha s ni kt ca lysozyme cng -adrenergic. Tn hiu (signal) truyn t th th trn (7-helix receptor) n mt protein ni bo c gi l G-protein, do vy nhm th th ny cn c gi l G-Protein-Coupled Receptors (GPCR). C khong 800 GPCR c tm thy ngi.

GPCR c nh hp (dimerize) hoc oligomer ha to thnh mt phc hp ni mng. Ligand bm vo th th s truyn tin i vo cu trc ny v sinh ra cc tc ng tng ng c trng cho tng loi th th. C nhiu loi GPCR-interacting proteins (GIPs) iu ha chc nng ca th th. Nhng tc ng ny bao gm: Thay i i lc ca ligand. iu ha s nh hp hay oligomer ha ca th th. nh v v tr ca th th, bao gm c vic vn chuyn hay loi b chng t mng t bo. Kim sot s tng giao ca cc protein thng tin khc.

G-prtein l mt cu trc gm c 3 tiu n v, c gi l , v . G-protein kch hot qu trnh hnh thnh cAMP trong t bo c gi l stimulatory G-protein hay Gs vi tiu n v , gi l Gs. Khi Gs c hot ha, nh khi th th p ng vi epinephrine v glucagon. Th th -adrenergic c gi l GPCR hng epinephrine. Tiu n v ca G-protein bm vo GTP v thy phn n thnh GDP v Pi. Tiu n v v c lin kt cng ha tr vi neo lipid (lipid anchors), do vy bm dnh v c nh G-protein vo mt trong mng t bo. Adenylate cyclase (AC) l mt protein xuyn mng, vng hng v pha bo tng c gi l vng thy phn (catalytic site).

Hnh 7.8: Minh ha rt gn qu trnh hot ng ca G-protein. Trnh t cc s kin xy ra khi hormone kch hot tn hiu cAMP:

Lc u, tiu n v ca G-protein ni kt vi GDP v ba tiu n v , v thng nht vi nhau v 2 tiu n v v c ch G. Khi hormone bm dnh vo th th GPCR, s c mt s bin i lp th xy ra v thng tin s truyn ti G-protein. Vng kt ni nucleotide (nucleotide-binding site) tiu n v tr nn nhy cm hn vi bo tng v v nng GTP ca bo tng tao hn hn GDP nn n gii phng GDP ni kt vi GTP. Qu trnh ny gi l GDP-GTP exchange. S thay i din ra trn dn n s bin i cu trc ca G v n ri phc hp lin kt vi Adenylate cyclase. Adenylate cyclase c hot ha bi G-GTP sau thc hin phn ng tng hp thy phn to ra cAMP. Protein kinase A (cAMP-Dependent protein kinase) thy phn v chuyn nhm phosphate ca ATP n nhm serine hoc nhm threonine ca mt vi protein ni bo v kch hot hot ng ca chng.

S ngt tn hiu (turn off of the signal): G thy phn GTP thnh GDP v Pi (GTPase). S tn ti ca GDP trn G khin n ti ni kt vi tiu phn v bt hot do vy adenylate cyclase khng cn hot ng na. Phosphodiesterase thy phn cAMP thnh AMP. Th th t lit (receptor desensitization) bin i cu trc khc vi cu hnh ca hormone. Trong mt s trng hp, receptor hot ha c phosphoryl ha thng qua G-protein receptor kinase. Th th c phosphoryl ha s ni kt vi -arrestin. Sau , -arrestn iu hnh qu trnh loi b receptor t mng t bo vo bo tng bi hin tng nhp bo nh tc ng gin tip ca clathrin (clathrin-mediated). -arrestin cng ni kt vi phosphodiesterase ni bo, mang enzyme ny n gn ni sn xut cAMP, gp phn lm ngng dng phn t tn hiu. Protein phosphatase thy phn loi nhm phosphate ca n. (trc n c phosphate ha bi protein kinase A).

S khuch i tn hiu (signal amplification) l mt cng on rt quan trng trong dng thc tn hiu. C th: Mt phn t hormone c th dn n qu trnh to thnh nhiu phn t cAMP. Mi tiu n v xc tc ca protein kinase A xc tc phosphoryl ha nhiu protein trong sut qu trnh tn ti ca cAMP.

Cc th ng phn khc ca G (different isoforms of G) c nhng cch truyn tin khc nhau. V d nh stimulatory Gs gn vi GTP th hot ha adenylate cyclase nhng khi inhibitory Gi gn vi GTP th n li bt hot enzyme ny. Do vy c th thy Gi c vai tr quan trng trong vic chuyn ngc li GDP thnh GTP v sau li hot ha Gs.

Hnh 7.9: M t s ADP-ribosyl ha. Phc hp G c gii phng khi Gni kt vi GTP dn n vic hot ha hay c ch mt vi loi protein khc. V d, G c ch mt vi th ng phn ca adenylate cyclase (isoform) gp phn ngt dng tn hiu vo t bo, ngng biu hin enzyme. c t t (cholera toxin) xc tc ng ha tr s bin th ca Gs. ADP-ribose c chuyn t NAD+ thnh arginine ti vng hot ng ca GTPase ca Gs. S ADP-ribosyl ha ngn s thy phn ca GTP (prevents GTP hydrolysis) bi Gs. Do vy stimulatory G-protein c hot ha lu di. Pertussis toxin (whooping cough disease) xc tc s ADP-ribosyl ti cysteine ca Gi lm n khng th chuyn GDP thnh GTP. Con ng inhibitory b kha (The inhibitory pathway is blocked).

S ADP-ribosyl l c ch chung iu ha hot ng ca protein, t sinh vt nhn s (prokaryotes) n sinh vt nhn thc (eukaryotes), k c ng vt c v.