Lecture 17. Terpenes and Cell Signaling, Part 1.
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| Teil | 17 | |
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| Identifikatoren | 10.5446/18876 (DOI) | |
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TriterpeneChemische ForschungMethyliodidKalisalzeHydroxybuttersäure <gamma->AmalgamISO-Komplex-HeilweiseMagmaAmmoniumverbindungenTerpeneEnzymDiphosphatePräkursorTerpeneZelleBukett <Wein>FlüchtigkeitQuellgebietChemische VerbindungenOktanzahlAktivierungsenergieStoffwechselwegErdrutschEnzymSetzen <Verfahrenstechnik>DiphosphateMolekülISO-Komplex-HeilweiseCytologieCyclische VerbindungenReaktivitätLimonenNaturstoffOrganisches MolekülIsoprenAdditionsverbindungenBET-MethodeFarbenindustrieZellfusionTaxolGradingAzokupplungFruchtmarkSynthasenQuerprofilMultiproteinkomplexTopizitätStrippenTerpenoideUntereinheitMethylgruppeCarbokationAktivität <Konzentration>ParfümeurTandem-ReaktionElektronische ZigaretteReglersubstanzExplosionChemieanlageChemische ForschungKohlenwasserstoffeVSEPR-ModellPosttranslationale ÄnderungWursthülleThermoformenGesteinsbildungPeriodatePhosphatePyrogallolPharmazieFunktionelle GruppeSchussverletzungChemische ReaktionSubstrat <Boden>Aktives ZentrumKomplexbildnerVorlesung/Konferenz
10:18
Alkohole <tertiär->TerpeneChemische ForschungDiphosphatePolypropylenGeraniolNatriumhydrogencarbonatVerstümmelungAmalgamHydroxybuttersäure <gamma->PrenylgruppeProteineMembranproteineVimentinBiomembranProlinSildenafilGalactoseDiphosphonateApatitHydroxylgruppePhosphateDrogeSymptomatologieAmmoniakQuellgebietWursthülleTerpeneIonenbindungEnzyminhibitorZelleChemische ForschungPrenylgruppeEnzymOsteoklastZusatzstoffMembranproteineCarbokationFleischerChemische ReaktionIsoprenChemische VerbindungenDoppelbindungAktives ZentrumOrganisches MolekülGummiPharmazieSynthasenPlasmamembranChemischer ProzessCarbanionBiomembranKohlenstofffaserSetzen <Verfahrenstechnik>CytologieDiphosphateTopizitätPhosphonsäureBiochemikerinLactitolBindungsenergieEthanThiolgruppeFarnesyltransferase <Farnesyltranstransferase>SyntheseölQuerprofilFunktionelle GruppeTerpenoideAzokupplungPentapeptideKlinische ChemieKrebsforschungOsteoblastCysteinDiphosphonateGangart <Erzlagerstätte>AllylverbindungenProteinkinase AFunkeKaschierenBukett <Wein>SchmierfettTryptophanDrogeMesomerieSekundärstrukturChemische StrukturFülle <Speise>Aktivität <Konzentration>Advanced glycosylation end productsOberflächenchemiePosttranslationale ÄnderungProteinbindungPhenylalaninTephraTrockenmilchSynthesekautschukStratotypInselEisflächeSingle electron transferThermisches KrackenPotenz <Homöopathie>Memory-EffektMetallBodenschutzIsoliergasElektrostahlPhosphorComputeranimationVorlesung/Konferenz
20:16
DiphosphonateChemische ForschungEstradiolBiosyntheseCyclische VerbindungenWundePolypropylenRetinoideOrnithinKernreaktionsanalyseCarbokationPenning-KäfigZellmigrationLactitolHydrideSekundärstrukturLaichgewässerAzokupplungWasserstoffFarbenindustrieFormylgruppeKonzentratAromatizitätCyclische VerbindungenMethylgruppePräkursorEnergiearmes LebensmittelKomplikationFeuerChemische ReaktionGenFleischerEpoxideBukett <Wein>EliminierungsreaktionEstradiolSetzen <Verfahrenstechnik>Beta-FaltblattGangart <Erzlagerstätte>NährstoffElektron <Legierung>GesundheitsstörungDiphosphateMultiproteinkomplexIonenbindungGletscherzungeWursthülleStoffwechselwegChemische VerbindungenCampherQuerprofilUntereinheitTandem-ReaktionBiosyntheseRetinoidePhytoenPharmazieEnzymOrangensaftSingulettzustandSynthasenTerpeneElektronentransferElektronische ZigaretteIsoprenTerminations-CodonPhthiseProtonenpumpenhemmerMassendichteBaseAusgangsgesteinSensePasteQuellgebietPhosphateInselChemische ForschungTellerseparatorBiologisches MaterialOktanzahlKohlenstofffaserAdvanced glycosylation end productsThermoformenTieftemperaturtechnikLandwirtinMetallPigmentKrankengeschichteComputeranimationVorlesung/Konferenz
30:13
TerpeneChemische ForschungCalcinierenKalisalzeBukett <Wein>IsoprenCyclische VerbindungenBleierzCopolymereMolekülSyntheseölMembranproteineDNS-SyntheseRNSGalactoseButterCarboxylierungSystemische Therapie <Pharmakologie>CobaltoxideTaxolMeeresspiegelMalerfarbeChemische ReaktionKohlenstofffaserReplikationPeriodateMolekülHydrophobe WechselwirkungEliminierungsreaktionMischanlageBaseChemische StrukturWursthülleFülle <Speise>f-ElementIonenbindungKohlenwasserstoffeAktivität <Konzentration>Aktives ZentrumSetzen <Verfahrenstechnik>QuellgebietAktivierung <Chemie>FarbenindustrieKrankengeschichteKonzentratChemische ForschungAcetyl-CoAWasserstoffReduktionsmittelMedikalisierungPräkursorTerpeneEnzymChemischer ProzessMembranproteineSekundärstrukturBiosyntheseWaldhonigGangart <Erzlagerstätte>BenetzungLSDPotenz <Homöopathie>TyrosinApothekerinNobelpreis für ChemieAlkoholische LösungCholesterinWasserstoffperoxidFlorentiner <Diamant>AromatizitätDoppelbindungSeitenketteGlutaminsäurePharmazeutische ChemieZelleMikrotubulusAzokupplungTiermodellPegel <Hydrologie>KoordinationszahlQuerprofilPentapeptideTiefseeOxideOktanzahlOrganisches MolekülGezeitenstromKrebsforschungPharmazieStockfischBlindversuchTopizitätCyclische VerbindungenCarbokationDeprotonierungAlkenePigmentAsparaginsäurePeroxideCarboxylateDepolymerisationPolymereInterkristalline KorrosionSynthasenHistidinPhenolErdrutschComputeranimation
40:10
Translation <Genetik>MolekülSyntheseölMembranproteineDNS-SyntheseRNSCopolymereChemische ForschungRNS-SyntheseEnzymAmalgamAlauneGalactoseHalbedelsteinSeltenerdmineralienZelleInsulinMucineRNS-SyntheseStoffwechselwegZelleSyntheseölLigandHomocysteinKernrezeptorChemieanlageChemische VerbindungenAgonistMolekülKonzentratReglersubstanzMembranproteineChemische ForschungPrenylgruppeFalle <Kohlenwasserstofflagerstätte>BiomembranTransformation <Genetik>AbbruchreaktionSetzen <Verfahrenstechnik>CrackAktivität <Konzentration>Bukett <Wein>Organisches MolekülPheromonMeeresspiegelElektron <Legierung>NaturstoffAcetylcholinrezeptorChemische ReaktionEinsames ElektronenpaarBiologisches MaterialSenseGangart <Erzlagerstätte>MaßanalyseChemischer ProzessHomogenisierenBindungsenergieOrbitalAtomorbitalSingulettzustandDNS-SyntheseMucineAktives ZentrumArzneimitteldosisAzokupplungScherfestigkeitWerkzeugstahlChemische StrukturZeitverschiebungWursthülleBranntweinQuerprofilPotenz <Homöopathie>KohleEnzymPeriodateVerhungernRingspannungKonvertierungWeibliche ToteMannoseComputeranimation
50:07
MucineZelleInsulinAmmoniumverbindungenChemische ForschungMagmaBenetzungEnzymSignaltransduktionRNS-SyntheseKernrezeptorSteroidstoffwechselInterferon <gamma->ZellwachstumTyrosinEpidermaler WachstumsfaktorSonnenschutzmittelGlutaminsäureIonenkanalStoffwechselwegMeteoritInterleukin 7MühleMultiproteinkomplexGinMeeresströmungZelleSystemische Therapie <Pharmakologie>Bukett <Wein>IdiotypSetzen <Verfahrenstechnik>Chemische VerbindungenStoffwechselwegWursthülleGenerikumSignaltransduktionBodenKernrezeptorRNS-SynthesePharmazieInselPigmentTyrosinPräkursorSenseOktanzahlAzokupplungKernproteineVerletzungMembranproteineWeibliche ToteInduktorFleischerMeeresspiegelGangart <Erzlagerstätte>Chemische ForschungHybridisierung <Chemie>MultiproteinkomplexMolekülPegel <Hydrologie>QuerprofilVerhungernSchmerzVerbrennungFrischfleischHeterodimereStimulansZutatGlucosetransportproteineCytologieAnalogaOrlistatChemische StrukturStromschnelleThermoformenFleischersatzMutagenAgonistWundeInsulinFarbenindustrieLigand <Biochemie>LigandHope <Diamant>SeleniteAllmendeParasympathikomimetikumIonenkanalG-Protein gekoppelte RezeptorenT-LymphozytFülle <Speise>AcetylcholinLactitolMucineComputeranimation
01:00:04
Chemische ForschungMolekülInhibitorPhthiseHydroxybuttersäure <gamma->MagnetisierbarkeitKernrezeptorLigand <Biochemie>AzokupplungChemischer ProzessLactitolKernrezeptorChemische ForschungSetzen <Verfahrenstechnik>FormylgruppeChemische VerbindungenMikrotubulusRNS-SyntheseInselTestosteronZusatzstoffSystemische Therapie <Pharmakologie>MolekülGangart <Erzlagerstätte>QuerprofilSenseIdiotypWursthülleFülle <Speise>StoffwechselwegKlinisches ExperimentMeeresspiegelTiermodellInhibitorMetabolitZelleAssemblyChromosomZelladhäsionWerkzeugstahlFluoreszenzfarbstoffFleischersatzEmbryoReglersubstanzEnergiearmes LebensmittelMembranproteineHärteBukett <Wein>Organisches MolekülCytologieZellteilungEnzyminhibitorBiologisches MaterialGraugussChromosomenaberrationPharmazieMolekülbibliothekEstranElektrolytische DissoziationDNS-SyntheseHydrophobe WechselwirkungBindungsenergieBlauschimmelkäseAdvanced glycosylation end productsLagerungPotenz <Homöopathie>KinesinLigand <Biochemie>MitoseFarbenindustrieBaustahlCalcitriolTaxolAlkoholische LösungComputeranimation
01:10:01
Chemische ForschungEstranStoffwechselwegBiomembranZelleKernrezeptorElektrolytische DissoziationVOC <Ökologische Chemie>CytoplasmaRNS-SyntheseBlindversuchLigandBindungsenergieLSDMagdMagmaDNS-SyntheseAmalgamArzneimittelPhytosterineSteroidstoffwechselGalactoseLigand <Biochemie>HomocysteinEnzymBiosyntheseVitaminMolekülSchmierfettZellwachstumChemische StrukturGenexpressionAlphaspektroskopieSteroidstoffwechselÖstrogenrezeptorQuellgebietTyrosinSojamilchPlasmamembranVitaminKernproteineKernrezeptorRNS-SyntheseTranskriptionsfaktorEnzymNatriumchloridNatriumiodidUltraviolettspektrumChemische VerbindungenBleitetraethylAntirheumatikumTriiodothyroninVitamin D3Energiearmes LebensmittelHelix <alpha->KochsalzIodWursthülleKonzentratSterblichkeitPharmazieAbleitung <Bioelektrizität>SekundärstrukturEstranZelleLSDEstradiolIodideChemische ReaktionDerivateMeeresspiegelEntzündungMembranproteinePräkursorEstronElektron <Legierung>GesundheitsstörungHydroxybuttersäure <gamma->Gangart <Erzlagerstätte>BindungsenergieHämatitStoffwechselLigandArgininDNS-SyntheseApothekerinReglersubstanzIdiotypErdrutschVerhungernCalcitriolBeta-FaltblattDibutylphosphatKlinisches ExperimentGasphaseHydroxyethylcellulosenDomäne <Biochemie>GenCholesterinTherapietreueSenseCytoplasmaFeuerTeststreifenFormylgruppeHydrocortisonTumorKomplikationOffener LeserahmenSetzen <Verfahrenstechnik>GrundumsatzOvulationshemmerChromosomenkondensationStrahlenschadenElektrolytische DissoziationHeterodimere
01:19:58
ZelleSetzen <Verfahrenstechnik>Vorlesung/KonferenzComputeranimation
Transkript: Englisch(automatisch erzeugt)
00:04
OK, we're back, sorry, a little slow this morning. We're going to finish up terpenes today and then we're on to cell signaling which is Chapter 9, the last chapter in the book. This is one of my all-time favorite Botticellis and that's saying a lot because I'm a big Botticelli fan.
00:22
And I want to present you with a mystery here which if you look closely is the color of the hair of these women versus the sky over here. So, anyway, a little bit of a mystery. We'll explain more very shortly.
00:42
OK, take a quick look at Chapter 9, just skim through it. It has a ton of really useful information especially if you go on in biology or anything bio-related. The goal of Chapter 9 is to orient you in this enormous and daunting sea of biological literature.
01:01
When you open up Science, Nature, or Cell, it seems like every new paper tries to redefine cell signaling and adds an additional layer of complexity. And the truth is as a chemist, this is the exact opposite of the way we like to think. Chemistry, we like to have a few very simple rules
01:20
that we can use to understand all of cell biology and all of, or sorry, all of chemical reactivity. Biology seems to be going in a divergent direction but it needn't be that complicated. And so the goal of Chapter 9 is to simplify all of that daunting complexity, strip it down into a few essential elements
01:40
that if you learn, then you can understand anything that you encounter in the area of cell signaling. And I'll be explaining that very shortly today. OK, proposals, they're coming up. They're due in two days, 48 hours. Again, attach a self-addressed stamped envelope. If you want comments back, if you don't want comments back, that's quite all right. If it's essential though that you follow instructions,
02:03
upload to turnitin.com as before. And if you're done at this point, you're wondering how you could possibly improve, try to write more concisely. So, concision is an essential element of good writing. The more concisely, the more precisely you write, the fewer number of words, the better.
02:21
I told you you can use up to 10 pages but if you use 5 and you're very effective at arguing, so much the better. OK, you'll be more effective as a proposal writer and as someone being persuasive if you can use fewer words. Fewer words is always better than more words. OK, any questions about the proposal?
02:41
This is your last shot. Last chance for questions. OK, let's start with you Sergio. Yeah? I wasn't too sure but I think it's said Thursday but the date said March 15th, so I. Oh, on the turnitin.com website? Oh, on the.
03:01
OK, well it really is this Thursday. Yeah, I assure you it's Thursday. I will take another look but honestly, I'm going to hold you to Thursday. So, OK, but thanks for the heads up. And then a question in the back?
03:21
No, no, no. And thank you for asking. OK, it's a completely different format than the journal article report. Actually, at the bottom of the instructions posted to the website, I tell you exactly what kind of fonts and margins I'm looking for. So, what I'm looking for actually is double space,
03:41
12-point, 1-inch margins, but I think it's all specified on the sort of towards the end of the instructions. OK, but don't follow the journal article report. That's a different assignment. For this assignment, I'm looking for a proposal. It has different sections, et cetera. So, it sounds like you're going to be busy.
04:00
Other questions? All right, good luck. I cannot wait to read your original and creative ideas. This is really one of the most fun things for me to do is actually read about these clever schemes. And I'll probably have them back for you within a week. So, that one I can return pretty quickly.
04:22
All right, oh, one last announcement. I don't think I have a slide about this one but I should announce it. Please fill it, please rate the class, evaluate the class through EEE. This is especially important if there's a chance that I might be writing a letter of recommendation for you. When I write letters of recommendation, I always type in the ID number
04:41
so that I can get a complete, I type in the ID number and the email addresses. And I do this just so I can get a complete record of all the grades. And something else that pops up is whether or not the student actually provided feedback. And it's randomized. The feedback is stripped from the ID. But it does give me a list of all the names of everyone
05:03
who provided information. And so, it also provides me a second list of people who didn't provide feedback. And my feeling is if I'm going to spend an hour or so working on your behalf to help you get into dental school or whatever, you could spend 10 minutes working on my behalf to help make the class a little bit better
05:21
for next generation. OK. So, if you want a letter of recommendation for me, be sure to fill out those evaluation forms. Any other questions about any announcements, things like that, where we're going? All right. We're down to the last two lectures. We have so much to talk about. So, why don't we hop right in.
05:43
OK. Oh, sorry. Some more announcements. I will have office hours tomorrow. This is your final, all bets are off time to ask me anything about the proposal. I imagine at that point, you're not asking me really crucial questions. I'm hoping that at this point, your ideas are pretty well set.
06:01
We will have discussion sections this week. And the discussion sections will again have the format of office hours so you can come and have your questions answered. Krithika has been sick for the last couple of days but I'm hoping she'll be back today. Get well soon, Krithika. Her office hours actually are today. She tells me that she's going to be there.
06:21
Miriam has office hours. Yeah. Could you move this to make it actually useful? That would be great. Thank you. Thanks. Thanks, Miriam. OK. Anyway, let's last announce this. All right. So, what we saw last time is that the terpene class of hydrocarbons that are naturally synthesized
06:44
and they are astonishing in their diversity and their biological activity. I should mention to you that these are the compounds that make perfumes smell good, that make flavors taste good. These are the compounds of life that make life worth living in a way, OK?
07:01
These are the things that we sense and we enjoy sensing. So, these are all assembled from isoprenes and head to tail fashion. And I'm actually going to start this today's lecture by picking this topic up again and looking at a little greater detail. And we talked about how these linear isoprenoid
07:20
pyrophosphates such as farticell diphosphate, geranil-geranil diphosphate, et cetera, can be cyclized by terpene synthases. And the terpene synthase folds it up and then hits it with the cyclization reaction which was a carbocation cascade that leads to these complex natural products.
07:41
Today, we're going to look at the actual carbocations as they go flying through the air, as they go on to make really complex natural products. And this is, I think, one of those objects of wonder that truly amazed me about the natural world. And then, we also discussed very briefly that terpenes are often oxidized
08:01
after the cyclization step. So, after cyclization, there was a series of tailoring enzymes that then took the skeleton of the hydrocarbon and modified, modified, modified until in the end, you ended up with this very bioactive compound such as taxol which is used routinely in anti-cancer therapies.
08:23
Terpene synthases, as we discussed, offer remarkable control over regio and stereospecificity. That's how you can have one enzyme that gives you a particular ring fusion, say a 6-6 fusion versus a 7-5 or even a spirofuse where the two rings were at right angles to each other.
08:42
This is truly amazing chemistry and it requires a catalyst that has been, that has evolved to have real control. The catalyst really exerts control over this regio and stereospecificity. OK. I want to start with this idea of head to tail fusion.
09:02
I kind of presented this without really describing it in great detail. And I want to go back over it just very briefly. This is a familiar molecule to us, the limonene. This is derived from two isoprene subunits. And if we call this sort of, this top part
09:20
with the two methyl groups, the head, these are fit together in head to tail fashion over here. And then I guess this is kind of tail to middle fashion on this side. But even before the chemistry that I'm showing you was defined, organic chemists have recognized that terpenes always seem to follow this head to tail rule.
09:42
And this is actually a pretty powerful way of figuring out how these things are chemically synthesized or synthesized not necessarily chemically in a laboratory but synthesized by microorganisms and plants that have these terpene synthase pathways. So, the way this works is there's the joining
10:03
that we talked about last time between the heads and the tails. And then we have a X leaving group over here. This is our pyrophosphate. That leaves and that sets you up for a cyclization to give you this limonene compound. And we'll look at it in greater detail.
10:20
But in short, this is going to set the stereochemistry. It's going to set that this carbon double bond is going to attack over here, et cetera. Whoops. OK. So, very briefly, head to tail emerges from how these things are synthesized. So, this tail over here at the pyrophosphate, the DMAPP,
10:40
the dimethyl allyl pyrophosphate has this good leaving group. Good leaving group steps out the door, setting us up with this carbocation and then the head of the IPP, the isopotenyl pyrophosphate can attack. That gives you naturally, this is the tail over here, head over here, so head to tail joining.
11:01
And that happens every time. OK. There's one really important exception to this. But for the most part, these things are held together and attached together in head to tail fashion. And so, this gives us a really important rule for dissecting how these things are built. Yeah. Yeah.
11:22
So, the primary carbocation? Yes. It's very unstable. Yeah. But it's in resonance with this allylic position to give you a tertiary carbocation. But, only the primary is reactive. The tertiary over here is held distant from this nucleophilic olefin over here.
11:41
So, because it's held away, it's not available for the reaction. Only the primary carbocation and remember, it's resonance. So, in the resonance structure, you're going tertiary, primary, tertiary, primary, tertiary, primary. But the primary gets the reaction and then Le Chatelier's principle drives it on to completion. Okay. So, that kind of sweeps it in one direction.
12:03
And I'll be honest, these, this class of compounds seems to ignore many of the rules that you've learned about in Chem 51 and sophomore organic chemistry where we learned about stability of carbocations and how dominant the stability of a tertiary carbocation is versus secondary or primary.
12:23
And in this class of compounds and this class of synthases, we find that the enzymes themselves are capable of overcoming those biases. And they overcome it by stabilizing preferentially one carbocation versus another. And I'll show you, I'll briefly extemporize to imagine
12:44
that the, or speculate that the active site would have something hovering right above this primary carbocation to give it a little extra stability. For example, you could imagine an aromatic functionality up here, a phenylalanine, a tryptophan that would be precisely poised
13:01
to do a cation pie stacking event and therefore stabilize the primary carbocation preferentially over the much more beguiling tertiary carbocation that otherwise would be the dominant more stable carbocation. And I thank you for asking because it's one of the really intriguing aspects of this class of compounds.
13:22
And it blows me away every time. OK. So, when we look at the structures that I showed you last time, notice that these are all joined together in head to tail fashion. So, we have tail, head, tail, head, tail, head, tail, head, tail, head, tail, head, tail, head. And this can go on for a really long time
13:41
like N, N being a thousand in the case of natural rubber. But they're all joined head to tail, head to tail, one after another. And they're all joined in that fashion without, you know, scrambling because the enzyme that forms these bonds is only set up to do a head to tail joining. And in synthetic rubber, chemists try to emulate
14:04
that kind of head to tail fashion type of joining as well. And so, in synthetic rubber, this head to tail joining is set into place by starting with some sort of head-based nucleophile.
14:22
So, this is a lithium, a lithiate. This is basically a carbanion. Carbanion attacks the tail of isoprene that sets you up with a new carbanion on this new head carbon. And then that sets you up to attack another tail, et cetera. OK. So, this actually works really, really well.
14:42
All right. Let's see. All right. I want to pick up our discussion and show you a different aspect of terpene chemistry. And the different aspect kind of neatly leads us towards cell signaling which is the final topic of this class.
15:01
It turns out that the localization of proteins in the cell is vitally important to dictating their function. In other words, you could have this really, really active enzyme that can turn on all kinds of things in the cell, but if it's not available, if it's somehow squirreled away somewhere in the cell,
15:22
then it won't have any effect on cell signaling. So, getting the protein to the correct place is a really important challenge in biochemistry and chemical and cell biology. And there's a couple of different ways that proteins are scooted to the correct spot in the cell. One way, for example,
15:41
or short peptide tags that act as zip codes to direct proteins to specific areas within the cell. Another way is for proteins to be tagged with isoprenoid derived, isoprene derived tails, okay, such as these farnesial tails that are added
16:01
on to the protein Ras, okay, where Ras is the GTP is. Okay. So, here's the protein over here. It has a biolate. It has a cysteine thiol. Recall the pKa cysteine thiol sets you up to have a thiolate at pH 7 quite readily. This thiol can then attack farnesial pyrophosphate
16:24
as catalyzed by farnesial transferase. And this will give you a farnesial attached to the thiol. Okay. So, now, you have this greasy part over here. Where do you think this thing gets localized to?
16:44
Okay. We have a little bit of greasy tail. Yeah. Membrane. Indeed, right. The membrane is a big greasy space as well. So, this acts as a greasy spike that drives this, that drives right into the membrane
17:01
and anchors the protein permanently on that plasma membrane. And that has important consequences. I'm skipping over some other steps. There's some other modifications that take place as well. But this basically takes RAS and confines it to a two-dimensional surface on the upper half of the,
17:21
or a two-dimensional surface on the barrier of the cell. And that kind of confinement is really important for its activity. So, because RAS plays a key role in cancer-based cell signaling, there have been attempts to derail this process and inhibit farnesyltransferase.
17:42
So, this farnesyltransferase is an important target for anti-cancer therapeutics. And unfortunately, none of those have worked out in the clinic. What ends up happening is they're so nonspecific for RAS getting farnesylated versus other proteins getting farnesylated that they have not been very effective in the clinic, unfortunately.
18:01
Because otherwise, it looked like a really promising approach. Okay. So, if we can disrupt this type of localization, then we'd have a way of dealing with cell signaling in many different kinds of cells. And here's another example. This is an example from the cell signaling of osteoclasts.
18:23
So, osteoblasts and osteoclasts are two types of cells that control the buildup and breakdown of bone tissue. So, the bones, the stuff that your bones are made out of. And this becomes especially important as people age. Their bones tend to get more brittle.
18:41
And so, it becomes really important to inhibit osteoclasts which are breaking down the bones. And so, one way of approaching that is to inhibit farnesyl diposphate synthase. So, if you inhibit this enzyme here, then you'd be preventing this farnesylation from taking,
19:03
this farnesyl diposphate from farnesylating key proteins and cell signaling and then killing the osteoclasts. Okay. So, the goal here is to inhibit this process here. And the strategy is to provide these kind of drugs.
19:21
These are called bisphosphonates. And these look, I guess, if you kind of look really squinty eyed at these, they kind of look like the substrate, right? I mean, you really have to squint. But you can definitely see that inherent to this phosphonate, right, where there's a carbon phosphorous bond, hence the name phosphonate, these bisphosphonates, because they have two of these, sort of look
19:42
like the biphosphates, the pyrophosphates of geranil diphosphate. And then some of these even have some greasy tails as well. Anyway, these compounds are prescribed in large quantities to women, especially as they get older, but also men as well,
20:01
depending on their circumstances. And in order to direct them directly to bone, there are often times they bind very strongly to hydroxyapatite, which is one of the key constituents for building bone and bone density.
20:22
Okay. So, this is an important osteoporosis treatment. Questions? All right. So, I want to return to where we left off last time in terms of complexity. And last time I showed you how the terpene synthases worked, but we didn't get a chance to really zoom in and look
20:40
at the carbocation cascades. And so, this seems like an appropriate point to pick things up. So, here's an example of different terpenes found in nature. These include the monoterpenes, which are C10.
21:00
So, all of these consist of two isoprene units joined in head-to-tail fashion. The sesquiterpenes, which are C15, again, all of these have three isoprenes. And then the diterpenes, C20s, that have four isoprene subunits. Okay. And again, if we spend time looking at this,
21:23
we'll find that all of these isoprene units are joined in head-to-tail fashion. Sometimes it gets complicated to find, but they're definitely in there. Okay. So, how do you get this rich structural diversity? How do you control this compound versus this compound?
21:41
Or even, you know, something like camphor versus this carine over here? And the answer is by controlling the mechanistic pathways of the cyclization processes that synthesize these compounds. And so, the terpene synthases that I described to you on Thursday are exceptional, really, at directing the outcome
22:04
of a really complicated series of reactions. And so, the one I'm going to start with is implicated in the biosynthesis of estrodiol. So, in this case, the enzyme, oxytocquiline linosterol cyclase, takes this epoxide and protonates it.
22:24
Okay. When it does that, the next step in here is a carbocation or a cyclization reaction where each one of these electrons hops, hops. So, it starts here, hops here, hops there. And in the end, you end up forming one, two, three, four,
22:44
four carbon-carbon bonds in one neat step, okay? I'm showing this to you as a concerted reaction, meaning all four bonds are formed in a smooth pathway. And this is one of the areas where chemists have been arguing for years
23:02
about whether all this takes place in one step or not. To my mind, the definitive experiment was done by E.J. Corey who showed pretty conclusively that actually there's sort of multiple intermediates that are formed during these carbocation and cyclization steps. And so, I'm showing you all four,
23:22
all five arrows hopping along at once. It probably doesn't happen exactly like that, okay? All right. All right. So, after this carbocation, after this cyclization cascade, this compound here, protosterol cation, undergoes a series of methyl transfer events.
23:43
Where hydrides and methyls, all the ones highlighted in pink over here, hop around and move and migrate. And by doing this, this gets you to eventually, linosterol, which then becomes the basis for,
24:01
so this becomes the basis then for all human steroids. Okay? So, this is a carbocation cyclization that all of you are doing even as we speak, okay? Let me just get you started with where these hydride shifts are going. So, after the cascade, you end up with a tertiary carbocation right here. So, you have this tertiary carbocation.
24:22
The first hydride shift is a 1, 2 hydride shift where this hydride in this H that's pink, hops over to here giving you a new tertiary carbocation. So, let's think about this for a moment. How might the enzyme preferentially stabilize one
24:41
carbocation intermediate over another and then drive this reaction onward? I'll give you a hint. I've already talked about this today. Chelsea? Indeed. All right. Well done, Chelsea.
25:00
So, you can imagine over here there might be an aromatic ring that's available to do a cation pi stacking to preferentially stabilize this carbocation over the other tertiary carbocation up here. All these things are directed. You can't have, you know, sort of random jumping around the ring.
25:20
All this is carefully controlled by the enzyme. And in the absence of the enzyme, what's more likely to happen is a beta elimination and none of these hydride migrations. But these hydride migrations are really profound, right? Over here, there used to be a methyl group over here. It's now hopped over to here. The methyl group that was over here is hopped over to here.
25:41
The hydrogen over here is hopped over to here. The hydrogen that was here hopped over to there. So, it's like a whole series of one, two methyl and hydride shifts where everything in the ring seems to be on fire in some way and capable of migrating and moving over. And it's like a game of musical chairs
26:02
where the final stop, the final carbocation is simply the most preferentially stabilized, likely as Chelsea pointed out through cation pie stacking type interactions. Make sense? All right.
26:21
Humans, unfortunately, cannot synthesize retinoids. And this is a really important, really a big nutrition issue in developing countries. So, the retinoids result from an unusual, tail-to-tail joining event, okay? So, here's geronyl geronyl diphosphate
26:42
and a second geronyl geronyl diphosphate. And check this out. They're going through an unusual tail-to-tail joining event, okay? That almost never happens. It happens in these super long geronyl geronyl diphosphate cases. But for the most part, this is rare, right? I mean, every other isoprene in here is joined
27:02
in head-to-tail fashion. And now, here's two tails that are coming together as catalyzed by phytoene synthase. Okay? There's a couple of more enzymes that go through here. And then, boom, you get down to the familiar beta-carotene of carrots, right? That's that nice orange color that we crave so much.
27:22
That color is essential. It's not just that it's colored orange and makes, you know, I was going to make a joke, but it would have been inappropriate. But it's useful because it plays important roles in human biology. And so, for example, if you have a diet that's missing this
27:43
beta-carotene, you end up with all kinds of unusual diseases that aren't really seen in developed countries like our own, but are definitely important in developing countries. And so, one of the challenges is trying to come up with diets that meet these nutritional requirements, okay?
28:01
So, for example, if you're on a rice-dependent diet, one way to do this would be to insert the genes that synthesize beta-carotene into rice resulting in nice orange-colored rice. It's called golden rice. And using that as a way of providing the nutritional requirements for the people in those countries.
28:21
This is really important. This is really something that I think all of us should be on the front lines working on. This is the kind of thing that makes a really big difference in the world. Okay, so a less weighty issue, but one that really surprised me when I first encountered it. This is a more trivial application.
28:42
It turns out that farmed salmon is farmed in a way to ensure that you can actually dial in the color of the salmon that results. And farmers, fish farmers, do this by feeding the salmon these precursors
29:01
to this astaxanthine pigment, okay? So you could basically with this salmon face, this salmo fan over here, you can dial in exactly the color of salmon that is going to be most appealing to the market. And if you go down to Albertsons, you're shaking your head, but it's true. You go down to Albertsons,
29:21
that salmon is a particular shade of pink that's going to best appeal to your, the Irvine consumer, okay? And it's all totally controlled by simply feeding in the right precursor chemicals so that in the end the fish have this astaxanthine. This is necessary because wild salmon will eat lots of shrimp
29:43
that naturally make their own astaxanthine. And the shrimp and the salmon that are farmed in big pens don't encounter shrimp as much. And so end up with a very different color. You wouldn't want to eat the non-pink salmon, okay?
30:00
It wouldn't be appealing. So anyway, same principle as the beta-carotene, similar chemistry as well. And then again, you just dial in using different, you know, dial in the final color based upon different concentrations of these precursors. Okay, so I'm going to switch gears now. I've been talking to you mainly
30:20
about human examples of terpenes. I want to talk to you just very briefly about the non-human examples. This is some of my all-time favorite paintings anywhere. These were done in the 15th century by the great Sandro Botticelli. And they're truly sublime, okay?
30:41
I mean, this is 15th century and these look astonishingly modern even today. I mean, they're just exquisite portraits of, in this case, this is Venus, the birth of Venus. And this case, this is the prime of era that I showed on the very first slide today. Okay, so even the best scientists like to take vacations.
31:05
And the great Conrad Block, winner of the Nobel Prize in chemistry for a lot of the chemistry I've been showing you today dealing with hydrocarbons, working out things like HMG-CoA reductase, working out a lot of the synthesis of cholesterol
31:20
that I've been showing over the last two lectures. That was all worked out by the great Conrad Block. Okay, and so he's on vacation in Florence, Italy, and he's looking at these paintings in real life for the first time. And the extraordinary thing that he noticed was that all of the models in these paintings and all these paintings have blonde-haired women in them.
31:44
Okay, and this is kind of a surprise to him. Okay, because if you're in Italy, most of the women have brunette hair. They have dark brown hair. And the thing that struck him was that these were painted in the 15th century, whereas hydrogen peroxide, which is the most common method
32:01
for colorizing hair from oxidizing hair pigments from brunette to blonde, was not invented until 1818. So the synthesis of hydrogen peroxide was invented by Thienard in 1818. So presumably, these women did not have access to large quantities of peroxide.
32:22
So an obvious question to a biochemist like Conrad Block is how did these women end up with blonde hair? Okay, or maybe the artist completely fabricated the whole blonde hair thing. Okay, and if you look at lots of other paintings from the same time period, it's very clear that blonde hair was something of a craze.
32:41
Okay, so Conrad's on vacation, and he likes mysteries. Oh, and by the way, he wrote a sublime book called Blondes and Venetian Paintings, Armadillos, and Other Medical Mysteries, highly recommended, great book. Anyway, so he's on vacation. He starts looking into this further, and what he finds is
33:01
that the women in that period would spend a large amount of time combing terpenes into their hairs. Okay, so they would have this kind of wet oily solution that they would comb into their hair, and then they would leave that kind of wet hair hanging out in the sun for long periods of time,
33:20
and what happens is a very inefficient chemical reaction that results in production of a tiny little bit of hydrogen peroxide, and over time, this will gradually lighten the hair color. Okay, so here's the way this works, starting with alpha-pinene and chlorophyll, you know, from the pine that this is derived from.
33:41
Chlorophyll is a triplet sensitizer that allows you to do basically a Diels-Alder reaction with the oxygen, and then here's the light over here. So this converts the singlet oxygen to the triplet state of oxygen, the triplet sensitizer, giving you an oxygen-oxygen double bond. Recall that oxygen is almost all oxygen
34:02
we're breathing is oxygen-oxygen single bond. Anyway, so this gives you an oxygen-oxygen double bond which sets you up for this Diels-Alder reaction, giving you a proxy intermediate that can then decompose to form this super-duper stable aromatic compound and releasing hydrogen peroxide.
34:20
Hydrogen peroxide can then dye the hair kind of blonde color, you know, this is not exactly, you know, blonde-blonde, but it's, you know, certainly honey-blonde, right? And, you know, the process might take a really long time, but the results, you know, were fashionable back then, okay? Questions about this? Anyone want to do this experiment for me?
34:41
I'd actually like to see this replicated, I've been reading about it for years, never seen it replicated. I understand it takes hours, might be fun. All right, let's talk about other cyclization reactions. The truth is things get really complicated if you get into this in greater detail.
35:02
Here, for example, is the taxodyne precursor to taxol and the carbocation cyclization that this goes through is pretty challenging, right? We're going to do, we're going to do a nucleophilic attack on a primary carbocation, we've seen that before,
35:23
we saw that on an earlier slide when we joined together the head to tail, isoprene precursors, there's then another cyclization to give us a tertiary carbocation and then a beta elimination over here. So this beta elimination gives us this for tisseline intermediate.
35:41
Fertisseline as an intermediate can then be protonated in a couple of different places, okay? And this is kind of an astonishing step, okay? So what we're going to see is a protonation of an olefin. And just take a moment to take a deep breath and appreciate that majestic unbelievableness, okay?
36:03
Because if you think about it, olefins are not a great basis, right? There's really no way that this step over here should take place, but it does. The enzyme has evolved a very effective acid that can protonate this olefin giving us a new tertiary
36:25
carbocation and setting us up for the last cyclization which then leads to a beta elimination to give us the taxodyne skeleton that becomes the core for forming taxol, okay? This is really astonishing chemistry. This is pretty remarkable stuff.
36:43
Okay, and then after you get to taxodyne over here you get to taxol. All right, anyone want to speculate on the identity of this acid over here? Okay, what kind of acids would you find in an active site? Someone not Chelsea.
37:00
Yes, Carl? What's that? Spartic acid, yeah, so that would be my first choice as well, aspartic acid, glutamic acid. The problem with those though is they have oxygen nucleophiles, right? Both of those carboxylic acids have oxygens and oxygens are really great nucleophiles. And the problem there is
37:21
that you have all these carbocations that are flying all over the place. And oxygen and carbocations don't mix. The oxygen is liable to go winging its way down to the carbocation giving you a permanent covalent intermediate. So instead when we look closely at the structures of terpene synthases that do this type of chemistry,
37:42
we find a completely different acid in an active site. It's not glutamic or aspartic acid. So that was an excellent choice. It was my first choice as well. It turns out it's not the case. Other choices, what other kind of acids would you find in an active site of an enzyme? And I'll, what's that?
38:00
Histidine. Yeah, yeah, so histidine, that's a good choice as well. Yeah, I can't argue with that one. All right, that's a good one. I'll tell you so that we'll just end this. Tyrosine is actually the choice. So tyrosine which has a phenol side chain actually acts as the acid to do this protonation step.
38:22
And that's completely counterintuitive because the PKA of phenol and tyrosine might be 10 or something like that. And here it is acting as an acid. The protonate is something that really doesn't want to get protonated. So you can imagine the neighboring side chains must in some way turbocharge the acidity of the phenol
38:41
of tyrosine making this reaction possible. This is pretty extraordinary chemistry. All right, any questions about terpenes, polyketides, that class of molecules, class of molecules we've been seeing?
39:02
Okay, questions about blondes and Venetian paintings? Chelsea? I was just wondering, so what is Taxol actually? Oh, okay, so Taxol is a really effective antibiotic that kills off the neighboring microbes. So microbes will synthesize this as a way of killing off their neighbors to compete.
39:21
But we chemists and medicinal chemists and pharmacists and physicians prize Taxol because it's very effective at inhibiting microtubule polymerization and depolymerization. And the fact is that it's very effective in the anti-cancer therapies. So it prevents cells from dividing and ends up killing breast cancer cells, for example.
39:46
All right, any other thoughts? All right, moving on. I want to switch gears now. We're now on Chapter 9, the final chapter, final topic for the book. And in a way I've kind of saved the best for last.
40:02
We really had to learn everything that we've learned this quarter so that we can understand at a systems level how cells really work. Okay, so we can now put together all of the information I've been giving you all quarter and now think about cells in a really chemical way.
40:20
Okay, and specifically we've been talking about the central dogma of biology and we're now ready to talk about how natural products and synthetic molecules can get in and disrupt key processes in different steps of the central dogma. And again, because natural products are synthesized
40:40
by enzymes, these enzymes down here are synthesizing things and we're going to be most interested in those that are interfering with transcription. Small molecules in general offer some really powerful abilities to control processes in the cell. For one thing, you have control over when they're applied.
41:02
Okay, so what this means then is you can have an organism that's grown to a certain stage of development and then you give it the chemical compound and you shut down a pathway at specifically a certain stage in its development and that allows you a really powerful tool to ask, all right, so at this stage
41:21
in development how does this protein pathway, how does this pathway actually impact the development of the organism? For that matter, you also have spatial control. So what I told you about was temporal control, spatial control meaning that you can dose in a specific type, a specific site in the organism.
41:44
For example, you might be interested in understanding I don't know just kidneys, okay, or let's just say just liver and how signaling by liver cells affects other organs. So you can imagine with small molecules you have a way then of just dosing in just the liver
42:01
and not the other pathways in the organism. So this temporal and spatial control is really powerful and it's one of the great strengths of using synthetic molecules as tools to dissect these pathways and so that's what we're going to be talking about for the next couple of days as we discuss small molecule control over the central dogma.
42:23
This has been done for thousands of years, okay. There's evidence of this that goes back to writings on Egyptian papyrus. You can actually find descriptions of small molecules that are isolated from plants like this one.
42:40
So for example, from this plant, this weird looking plant over here, you can actually isolate hyoscabene which is isolated again from the plant depicted over here. This is known as belladonna, nightshade, henbane, mandrake and it has the structure, this structure here.
43:01
Notice that its structure mimics the structure of acetylcholine, okay, and this is one you don't really have to squint at. These two look really similar to each other, especially when you draw them like this, okay, and then here's the structure of scopolamine, another related compound in this. And so these compounds act as agonists
43:21
for acetylcholine receptors and these compounds are found in really high concentrations in seeds and berries of these plants because it encourages animals to grab onto the seeds and berries and move them around thus spreading the plant and making it more successful as a Darwinian evolved creature,
43:44
okay, so we humans are also very big on psychedelic experiences and so we also prize these seeds and berries. I will tell you that you should definitely read the
44:01
warnings if you can start experimenting with these. These are really come down with very, very serious side effects and even death. But for centuries these have been used and they work again by targeting acetylcholine receptors which I believe we talked about in the context of paralysis, right.
44:20
So for example, witches who, you know, for centuries were thought to fly, they're probably flying metaphorically using compounds like these isolated from belladonna, nightshade and other plants, okay. And the straightforward way that they would do this is they take these seeds and put them in various membrane passages, right,
44:45
so up your nose, et cetera. And actually it works, they work surprisingly. There's a high concentration of these compounds in these things. Okay, in addition to humans wanting to spread these things, small molecules are used
45:00
by other animals to signal each other. So for example, the avocado seed moth shown here responds to this pherabone, this compound over here and this is actually a powerful way that we can use to trap the pest to attract it. This is a pheromone that it uses to attract its mates. Okay, so signaling by small molecules is used
45:23
for therapeutics, it's used by organisms to signal each other, it's used recreationally, it's used in all kinds of different contexts. And it's pretty much universal. Every organism that we find, even the most simple organisms, even something like E. coli use small molecules
45:41
to talk to each other. So this really is a universal mode of communication. And it's kind of astonishing that we humans actually use different modes for communication. We're not so dependent upon this. But your dog, your dog is very dependent on small molecule signaling as evidenced by the fact that it spends all of its time sniffing, right?
46:02
Your dog sees a rich universe of small molecules all around it when its sample sense in the air. Okay, before I get into how these small molecules work, we have to have a little chat about arrows. This is from experience, I know that this is one of the big hang ups that organic chemists have
46:22
when they start diving into cell signaling. Arrows in chemistry and arrows in biology have two fundamentally different meanings. Okay, in the chemistry world, we have arrows that indicate transformations from reactants going to products, often with some catalyst.
46:40
These arrows can be equilibrium arrows, they can be single point arrows indicating no return reaction. But this is a familiar arrow to us. For that matter, we're also very familiar with arrows that show electrons or overlap of orbitals from the highest occupied orbital,
47:02
the homo of this nucleophile lone pair to the lowest unoccupied molecular orbital of the electrophile over here. Okay, so these are arrows that we use conversationally all the time in this class and in other classes. And I've been using these since the very beginning of this class, I've used them today multiple times, they're totally understood.
47:24
Arrows in biology means something different, okay? First, when there is an arrow, the arrow means directly act, direct activation, okay? So for example, this arrow indicates that this ligand binds
47:41
to its receptor over here, which in turn binds to this farnesylated protein over here. Furthermore, if this farnesylated protein does some transformation, it's going to be acting as a catalyst. And the way we indicate it's a catalyst is the way we indicate it's a catalyst is a chemical transformation
48:00
by simply placing it in the center of the arrow, okay? Now, there's a whole bunch of other arrows that we find in biological communication. So for example, arrow that has a terminator at the end, I don't even know what you would call this.
48:20
Anyone want to take a crack at that? What would you call this end point one? All right, the perpendicular line, it indicates direct inhibition, okay? So this would be, so somehow this guy over here directly inhibits this protein over here. Oh, if it's a dotted line, it indicates indirect inhibition,
48:42
meaning maybe there's an intermediary. So this guy binds to this guy, which binds to this guy, which somehow inhibits this, okay? So dash lines indicate indirect effects and solid lines indicate direct effect. And then finally, there's the arrow that has the arm on it that indicates activates transcription, okay?
49:03
So all of these arrows, this is kind of the language of biology, they follow these conventions. They're really not that daunting if we take a moment to think about them. All right, within cell signaling, there's three scenarios. Oh, so we're all good on the arrows, arrow business, we don't have to talk about that anymore.
49:21
Okay, we're going to be using them, that's the convention we're going to follow. Within cell signaling, there's going to be three kinds of scenarios for cell signaling. And each of these scenarios evolve depending on the requirements of that type of cell. For example, on the left, you can imagine signal will arrive
49:41
and then the cell has to immediately respond. In that case, the cell will release something that it's pre-synthesized and no transcription is required. So the signal never reaches down to the level of transcription of DNA. An example of this, I'll show you on the next slide, it's the release of mucin by goblet cells.
50:02
Another type of signal is signal arrives, the cell then has to synthesize a bunch of new proteins and respond to that signal, transcription is required. If transcription is required, things slow down, okay? So this is a slow type of signal over here. For example, skin cells that have to fill in a wound, okay,
50:25
so you cut your finger with some paper or something like that, the skin cells nearby have to proliferate and fill in that injury and so this is not going to happen instantaneously, right? You know that it takes a couple of days for the paper cut to heal, right?
50:42
It doesn't happen instantaneously, doesn't have to happen instantaneously for that matter and so this is a slow type of cell signaling. Commonly, there's both a slow and a fast response. For example, in response to insulin, so the insulin arrives, the cell immediately starts responding using the glucose
51:02
transporters that we discussed when we talked about glycobiology a few weeks ago, but in addition, the cell has to respond to this insulin by synthesizing new proteins and having a slow response. So this hybrid is also a very common mode as well, okay?
51:21
So we're going to see these three different scenarios play out. The first scenario is this business about mucin. So for example, acetylcholine agonists, it is a stigmine stimulates intestinal goblet cells to spill out mucin in a rapid response, okay? So this is you, you're eating something
51:42
and you encounter something that's bitter like this physo stigmine and you have to immediately respond to that. You know, your stomach doesn't like bitter stuff and so the stomach and the intestines respond by secreting all this mucin. We've talked about the structure of mucin. It's an oligosaccharide and that helps smooth the
52:01
passageway and get rid of this bitter-tasting compound. Okay, so here's the goblet cells over here. They've already stored up all the mucin, okay? It's already over here. So as soon as this acetylcholine agonist arrives, you immediately start spewing out the mucin. It flows out into the intestines and it's already,
52:22
you know, been pre-synthesized. So no DNA transcription is required. You get an immediate response. Okay, now I'm also trying to systematize cell signaling. So also on the level of systematization,
52:41
cells can either be talking to each other, to different kinds of cells, or they can be talking to the same class of cells. And these two different classes of cell signaling are referred to as either autocrine or paracrine. Autocrine is communication between cells of the same type. Okay, so if those goblet cells are stimulating each other,
53:03
that would be autocrine. If, however, they're dependent on a different class of cells to stimulate them, that would be paracrine. Okay, make sense? Okay, now it turns out that within all of biology there's really only seven different types
53:21
of cell signaling. Okay, and I want to make this point. This is super duper important because when you pick up the literature in cell biology and signaling it's insanely confusing and complex. Cut through all that complexity. All that matters is that there are seven different types
53:41
of cell signaling. If you learn these seven different types of cell signaling you will understand all of the different kinds of pathways that are written about in the current literature. Okay, and one of the problems with, okay, so I'm starting to get off the track, I can't resist. Okay, so one of the things that makes this difficult is
54:01
when biologists write about their own pathways, they always describe how their pathway is unique and how it doesn't fit into the, nonsense. Okay, every single pathway can be fit into one of these seven categories over here. And as a chemist I want to know the seven simplest examples and then I want to be able to apply those seven simple examples
54:21
to every other example that's out there. And I promise you, you can do this. This will make sense of all of cell signaling. Okay, so all we have to do over the next couple days is simply learn about these different types of receptors and ligands and their cell signaling transduction pathways that modulate transcription and this is going to simplify
54:43
an otherwise ridiculous level of complexity. Okay, let's get started. This is, so this diagram is really basically all you have to know. This shows the seven different types of cell signaling.
55:00
Starting with the most common, nuclear receptors. So nuclear receptors are controlled by ligands that reach directly into the nucleus and turn on transcription. Hence the name nuclear receptors, okay? And I'll show you examples of each of these. Also at the top are two component pathways where you have a dimerization event up here
55:23
that eventually reaches down to transcription. And note again that these dashed lines indicates indirect effects. So in this case, the ligand is going to go all the way into the nucleus. In this case though, it's going to have a whole bunch of intermediaries that are going to have multiple steps
55:41
to eventually get to the nucleus. In addition, we have receptor tyrosine kinases, trimeric death receptors, G-protein couple receptors, ion channel receptors, and finally gas receptors. And from there, all of cell signaling can fit into these seven categories.
56:01
So all we have to do is learn these seven and then I hope everything else will make sense. Okay, sound like a plan? Let's get started. I'm going to start on the most common which are nuclear receptors and then we'll step systematically through each of these. Oh, okay, before I get to that,
56:20
I have to introduce a couple of more terms. The first of these are terms that are taken directly from the area of genetics. The terms are forward genetics versus reverse genetics. And the best example I can give you this are drosophila. So fruit flies naturally have red eyes, okay?
56:44
And if you radiate a whole population of fruit flies or you feed them some sort of chemical mutagen, you will eventually find fruit flies that have white eyes. Okay? They will have this white phenotype, okay? They look like this, red eye, white eye.
57:01
And then if you spend time looking at the different mutations that result in the white eye, you will figure out that actually this results from impaired mutations that prevent the pigment precursors that result in the red eye from getting
57:20
across the cell membranes, okay? So white eyes result from mutations that prevent those pigment precursors that eventually result in the red eyes over here, okay? So this approach is called forward genetics. You make a whole series of random mutations and then you look at the types of phenotypes that result
57:42
from those random mutations and use those different random mutations to dissect the biology that results in those phenotypes, okay? That's called forward genetics. You make mutations, you look for a response, you then understand how that response occurred, okay?
58:01
The opposite would be reverse genetics. Not exactly opposite but it's complementary. Before I get to reverse, any questions about forward genetics? Okay, so forward genetics, random mutations, hope for an interesting phenotype. This is a strategy that's worked well for over 100 years. Biologists have been doing this for a really long time.
58:22
Chemists have in the last couple of decades applied a, actually no, no, I would say this has been going on for a much longer time than that. Let's say at least five decades, maybe even longer. Chemists have been applying a chemical analog of forward genetics called forward chemical genetics
58:42
where you apply a bunch of different small molecules to introduce random changes and you hope to find an interesting phenotype, okay? So, maybe you have your fruit flies, you apply a bunch of different chemicals to fruit flies and you look for ones that give you white eyes, chemicals that result
59:00
in white eyes and then that identifies chemicals that are in some way preventing the pigments from reaching the eyes to give you the red color of the fruit flies, okay? Here's one example of this. This is a compound called rapamycin, isolated from Easter Island soil, so soil from Easter Islands out in the Pacific.
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This compound in the very center acts as the glue in a molecular sandwich between FK506 binding protein 12, has molecular weight of 12 kilodaltons and another compound called mammalian target of rapamycin, mTOR, okay? So, this is FKDP on the left in purple,
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mTOR on the right in green. And you could see that the rapamycin is acting as the meat in the sandwich to bring together this complex and the net result is once this complex forms, the rapamycin prevents amongst other things,
01:00:00
T cells from forming. And so, rapamycin is a really important immunosuppressor. I think we've talked about this before actually, maybe a couple of times in this class. This is a molecule that prevents the immune system from responding to say liver transplants, okay?
01:00:20
Okay, so again, this is called forward chemical genetics because we're going to use random compounds that are isolated from some soil from like a distant island. And then, we're going to look for ones that have interesting effects, in this case, the phenotype of suppressing the immune system. And then, we use the compound to study how these immune system pathways work.
01:00:44
So, rapamycin has been a really powerful compound for figure, for dissecting these pathways. And in recent years, it's also had an important effect, an important tool, it's been an important reagent in studying aging and diet and other types of pathways.
01:01:07
Okay, so small molecules offer control over otherwise unavailable phenotypes. And so, for this reason, they've come to the floor as really good power tools in cell biology.
01:01:22
So, for example, the classical genetics approach starts to break down when you get mutations that kill the organism, okay? So, for example, this focal adhesion kinase, if you make mutations to focal adhesion kinase, you end up with these, you know,
01:01:43
embryos that are hugely distorted and really incapable of living. Okay, so the classic genetic forward genetics approach, not going to work. So, instead, what you do is you identify a small molecule inhibitor of focal adhesion kinase and then
01:02:01
that offers a solution that inhibits focal adhesion kinase and gives you a way of studying this otherwise lethal phenotype. So, that way, you can dissect how the protein actually works, okay? All right, now, of course, doing this requires some way
01:02:22
to look for interesting phenotypes, right? We need some way to go out and identify in forward chemical genetics weird cellular phenomena. And so, chemists have been very clever about doing this. This is an experiment done by Tarun Kapur and Tim Mitrissen.
01:02:41
And what they did is they had a whole library of different compounds. Here's one compound shown here and they start with 16,000 compounds and in a plate that has a large number of wells, they look for some mitotic signature, okay, which has this dark blue color. Okay, so they set up an assay that looks
01:03:02
for a particular phenotype, dark blue, under that assay, okay? And then, they take the compounds that light up in this assay and look for the effect and look for the compounds that can stabilize microtubule formation. Okay, so earlier, we talked about Taxol. Here's a Taxol stabilizes microtubules.
01:03:22
Here's another set of compounds. Fifty-two of these destabilize microtubule formation. One stabilizes it and the compounds that are positive here then go on to fluorescence microscopy experiments. Over here, these are low throughput. Over here, these are high throughput.
01:03:41
Over here, you have 16,000 compounds. Down here, you have less than 100. Okay, staring at cells under a microscope, that's hard work. Okay, over here, just looking for the one dark spot out of, you know, thousands, tens of thousands of dark spots, that's easy. This step over here can be done by robots. This step over here can be done by robots as well,
01:04:02
but sometimes, the robots miss stuff. Sometimes, the imaging software misses really interesting effects. Okay, and what Kapoor and colleagues found is that this compound called monosterol has this really astonishing effect on the kinetic core
01:04:20
which is this assembly of chromosomes during cell division. Okay, so normally, the chromosomes line up kind of like on the, forgetting my football analogy, scrimmage, the line of scrimmage, sorry.
01:04:40
Okay, so everything lines up on this line of scrimmage during cell division and then these chromosomes are pulled apart into the two daughter cells. Monosterol explodes the whole thing. And so, instead of having chromosomes in the center, microtubules on the sides, instead, you end up with chromosomes on the outside and this crazy microtubules in the center
01:05:02
and that's absolutely fascinating. It turns out that this compound is an inhibitor of one of the motor proteins that drives the chromosomes and pushes and shoves the chromosomes into place during this process. And so, that gives you a really powerful tool for dissecting how cell division takes place.
01:05:23
Okay, so any questions about using small molecules in this? Yeah? Yeah, so the gist of this one is that they wanted to figure out if they could interfere with mitotic divisions. So they dyed the chromosomes blue and then checked a bunch of molecules to see
01:05:41
if any of them disrupted them. Exactly. And I'll be honest, they didn't expect to have, you know, something like this. Like they weren't looking for this pattern. They were just looking for weird patterns. In the same way that you'd look for white eyes or just weird colored creatures in the case of the fruit flies, the Drosophila.
01:06:02
You're just simply looking for something that's abnormal. And then you, once you find that abnormal thing, then you go in and use this as a tool to figure out how this process works and what proteins must be implicated. So this implicated a motor protein called kinesin, a particular type of kinesin in this process.
01:06:20
That otherwise, it wasn't clear exactly what it was doing. Sound good? Powerful tool? OK, yeah, question in the back? Yes. Yes. Well, if it's a dominant mutation,
01:06:41
then you cross-breed the dominance together and then you definitely see it. If it's recessive, then you'd see it in one out of every four, you know, daughters, one out of every four progeny. That's the genetics. And also, it's complicated because it also depends on how many mutations are required. It starts to get complicated quickly.
01:07:01
But my genetics friends could tell you all about that, OK? But it makes sense, right? That yes, you'd see those mutations coming through. Whereas, if you use a small molecule, you know, once the small molecule is metabolized, once it's hydrolyzed or whatever, oxidized, it's game over. Its effects disappear. And that too is powerful, right?
01:07:21
So you apply the small molecule and then it goes away and you could go back to wild type. And so you could see what it looks like if the small molecule is not there in exactly the same organism. OK. All right. So I want to get back to the seven different kinds. Now that we understand how small molecules are going to play important roles, I'm going to highlight the important
01:07:43
of the roles of those small molecules as we start looking at the seven different pathways for cell signaling. One of the most common of these cell signaling pathways are the nuclear hormone or the nuclear receptor based pathways.
01:08:02
There are some 28 nuclear receptors. Only eight have well identified known ligands as recently as a couple of years ago. OK. So these eight are depicted here. So some of these are familiar to us.
01:08:22
I believe we talked about the iodo, these iodo, these iodinated hormones earlier in the quarter. We talked earlier today about the process of synthesizing estrogen which eventually could be further modified
01:08:41
to give us estradiol. Here's testosterone over here. Here's calcitriol over here. These are all steroid receptors, right? All these guys up here are steroids. These guys down here, retinoic acid, iodo thyrene, these are other hormones. These hormones bind directly to nuclear receptors and then
01:09:02
as we'll see in a moment, the nuclear receptors directly carry the signals all the way to the DNA level. Note that when these things are bound, when these hormones over here are bound, they're completely engulfed by the protein. The protein throws a big old sloppy bear hug
01:09:21
around the steroid over here and it's completely buried. OK. So in this space filling model, you don't see any yellow. OK. It's completely covered up. The thing is heavily buried by the receptor. The receptor changes its confirmation upon binding to the hormone and that in turn allows it to do stuff
01:09:44
like get into the nucleus and start affecting transcription. Oh, by the way, I told you that these are the eight that are known, the other 20 sort of unknown. It's thought that those other 20 bind to various metabolites weekly, OK, meaning not very strongly.
01:10:02
And so, that binding is thought to be a little non-specific and a way of sampling many different states in the cell. So different steps along metabolism and other intermediate compounds as well. OK. So why don't we take a look. There are two modes of nuclear receptor signaling
01:10:20
that we need to learn about. One of these is illustrated by estrogen. So here's estrogen over here in the hypothalamus, in the hypothalamic neurons. In this case, the estrogen binds to the estrogen receptor. Estrogen receptor then dissociates from the heat shock protein and that frees
01:10:42
up the estrogen receptor to eventually make its way to the nucleus. OK. What you need to know about this is that estrogen receptor binds to the estrogen, to the steroid and then it gets all the way to the nucleus. It doesn't get to the nucleus before then because it's kind of covered up by these heat shock proteins. And furthermore, it has to bind to this carafirin beta,
01:11:05
carafirin beta molecule over here to make it through the nuclear pore and be imported into the nucleus. Once it's there, it can then trigger transcription of the gonadotropin receptor hormone gene.
01:11:21
OK. So this triggers the transcription and then in turn then allows the cells to respond to the estrogen. OK. Something else that's notable about this, notice that this has a homodimer. OK. These are two identical molecules of estrogen receptor alpha over here and then estrogen receptor over here
01:11:42
and they're both identical. Contrast that against the other mode of nuclear receptor signaling which involves heterodimerization. Same principle, the molecule diffuses. In this case, the molecule is retinoic acid. Structure is here.
01:12:01
Oh, and by the way, again, this is structure of estradiol. Estradiol, again, synthesized from lanosterol. Retinoic acid over here can directly sneak its way across the plasma membrane through the cytoplasm, through the nuclear membrane and get all the way over to this receptor for the retinoic acid.
01:12:23
So this is the retinoic acid receptor alpha. And this also binds to the RXR receptor and then the two of these get together and heterodimerize before leading to production of these homeodomain encoded proteins.
01:12:43
OK. So this leads to transcription and expression eventually of these HoxB proteins. OK. So in both cases, we see a similar effect. You have this greasy little hormone molecule. The greasy hormone molecule gets into through the plasma membrane, binds to the receptor and then the receptor sneaks into the nucleus
01:13:03
where it can cause transcription. OK. So in this case, the signaling molecule is taking a shortcut to get all the way to the nucleus. Sound good? OK. Let's look at a structure. So this is a structure of the PPAR gamma.
01:13:25
This G should be gamma binding to retinoic acid receptor alpha bound also to this ligand over here, rosiglitazone and retinoic acid in yellow up here and then also binding to DNA.
01:13:41
So this is an example again of heterodimerization, blue, purple, PPAR gamma, RXR alpha and then this heterodimer can interrogate the DNA using exactly the same sort of transcription factor event that we talked about when we talked
01:14:00
about proteins binding to DNA many weeks ago. In fact, maybe over a month ago. It seems like a really long time, earlier in the quarter. And again, notice that this has this alpha helix that could snugly fit into the major groove and interrogate the sequence. So this thing will then look for a particular sequence of DNA and then activate transcription
01:14:20
of a particular gene, a particular open reading frame of that DNA leading to the turning on of specific proteins, the production of specific proteins. Make sense? OK. So much to talk about here. And unfortunately, I just don't have time.
01:14:42
We're running out of time. Suffice it to say, chemists have spent a lot of time thinking about ways to control nuclear receptors. Because if you can, then you can dramatically control gene transcription and in turn, that could have dramatic consequences
01:15:02
for controlling the growth of cells, the mortality of cells, the differentiation of cells, et cetera. And this has been done classically for well over 50 years now. For example, isolated from the Barbosco yam,
01:15:23
this compound over here, check out this tuber. This thing is enormous, right? That's a whole tuber down here. This is what the yam tree looks like. It's just one monster tuber. You can isolate from this tuber, this compound Diosgenin. And from Diosgenin, you can make estrone.
01:15:41
You can make a whole bunch of other steroids. And you can imagine wanting to make this for birth control pills, for hydrocortisone, for anti-inflammation, prednisone, other inflammation effects, et cetera. OK. Nowadays, so back in the 50s and 60s, a whole series
01:16:04
of chemists, a whole generation of chemists went to Mexico where the Barbosco yam has been cultivated for centuries. They went to Mexico and they isolated directly out of these very high percentages of the precursors like this one for synthesizing these hormones.
01:16:22
Nowadays, we found that even though they're present at lower concentrations in soybeans, because soybeans are so grown so ubiquitously at such high levels in the United States that we can regularly, we can much more simply isolate
01:16:42
phytosteroids directly from soy. And so, the ultimate source nowadays of many of our steroids is from soy phytosteroids, OK, so rather than the Barbosco yam, which is too bad because the yam has a wonderful cultural heritage. Other non-steroidal ligands,
01:17:01
nuclear receptors include these iodo compounds, tetraiodothyronine and tetra triiodothyronine over here. The important thing to know about this is that both of these compounds are derived from tyrosine. This is iodinated tyrosine and these are important
01:17:21
for regulating the basal metabolic rate. And so, if your diet does not have enough iodine in it, you could run into all kinds of complications, OK, including goiter that's shown here. And I put the picture up because I suspect no one in this classroom has seen a case of goiter, right?
01:17:40
This is just to be totally common until all the salts in the country except for kosher salt was iodized. OK. So what this means is that there's a low concentration of sodium iodide mixed in with the sodium chloride. And it turns out that it doesn't require a very high concentration of iodine, iodide in the diet to get just enough
01:18:01
to synthesize naturally these iodinated tyrosine derivatives. All right, questions? Anything dealing with nuclear receptors? OK. One last thought, calcitriol, this compound over here requires UV light
01:18:23
for an electrocyclic ring opening reaction. So, you start with dehydrocholesterol. The compound does this very weird 6 pi electron electrocyclic ring opening. Electrons are going to bounce, bounce, bounce. And this gives you a ring opening. And then, there's a 1, 7 sigmatropic shift shown
01:18:45
by these arrows over here which you might have to go home and convince yourself of yourself. But long story short, this gives you vitamin D3. If you have a deficiency in vitamin D, this leads to rickets, the bow-legged phenomenon.
01:19:01
This is just one of the many manifestations of this really terrible disease. But again, the key is that in our diets, we get enough vitamin D3. This leads to liver enzymes and kidney enzymes that convert it to calcitriol. And then calcitriol binds directly to nuclear receptors.
01:19:25
OK. Final thought, last slide of the day. If for example, you have a mutation in the calcitriol receptor, the mutation of this arginine over here to leucine, you can chemically synthesize.
01:19:42
I'm talking like you do this in the laboratory and then, you know, give it to a pharmacist who gives it to a physician. You can have a chemically synthesized derivative of calcitriol that then fills in the mutation over here, binds the receptor and gives you normal cell signaling. OK. Well, why don't we stop here. When we come back next time, we'll be looking
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at the other six types of cell signaling.