Tuning cell-surface receptor signaling through structure-based ligand engineering

Tuning cell-surface receptor signaling through structure-based ligand engineering

Articles, Blog , , 0 Comments


WELCOME TO THE WEDNESDAY AFTERNOON LECTURE FOR THOSE IN MASUR AND THOSE WATCHING REMOTELY BY VIDEO. WE’RE GOING TO HAVE PRESENTATION FROM DR. GARCIA TALKING ABOUT TURNING CELL-SURFACE RECEPTOR SIGNALING THROUGH STRUCTURE-BASED LIGAND ENGINEERING: WNT/FRIZZLED AND TALKING ABOUT A COUPLE OF SYSTEMS, WNT/FRIZZLED BUT SOMETHING LESS FAMILIAR FOR THOSE US WHO HAVE HAVEN’T FOCUSED ON THAT PART OF CELL BIOLOGY. DR. GARCIA IS PROFESSOR MOLECULAR AND CELL PHYSIOLOGY AND STRUCTURAL BIOLOGY STANFORD UNIVERSITY SCHOOL OF MEDICINE AND INVESTIGATION AT HOWARD HUGHES MEDICAL INSTITUTE. HIS FACULTY CAREER HAS BEEN ALMOST ENTIRELY AT STANFORD WHERE HE CAME UP THROUGH THE RANKS AS ASSISTANT PROFESSOR THEN ASSOCIATE THEN INVESTIGATOR OF HHMI AND CORE PROFESSOR STARTING IN 2007 AND AN ELECTIVE PROFESSOR OF THE NATIONAL ACADEMY OF SCIENCES. HIS WORK HAS COVERED A NUMBER OF SYSTEMS BUT I’LL FOCUS ON THIS IDEA ON HOW LIGANDS AND RECEPTORS CAN INTERACT AND SOME OF THE STRUCTURES HE’S PRODUCED, MOST NOTABLY THOSE OF US WHO SAW PAPER ABOUT FRIZZLED HAS BEEN QUITE STUNNING. PERHAPS HE’LL TALK THE ABOUT THIS THUMB AND INDEX FINGER, IT’S REMARKABLE THREE DIMENSIONAL STRUCTURE. I LEARNED HE’S AN ULTRA MARATHONER AND A BANJO PLAYER. WE AGREED, THOSE THINGS ALWAYS GO TOGETHER. [LAUGHTER] IT’S WONDERFUL TO HAVE HIM HERE TODAY. PLEASE JOIN ME IN WELCOMING DR. CHRISTOPHER GARCIA. [APPLAUSE] [LOW AUDIO].>>THANK YOU FORKING HAVING ME HERE TODAY. IT’S A BIG DEAL, I KNOW [LOW AUDIO]. SORRY. EQUALLY PROFOUND PHILOSOPHICAL ENLIGHTENMENT AS THE DALAI LAMA SO I’LL TRY TO DO THAT. SO TODAY JUST TO GET STARTED, I’LL IT WILL YOU A LITTLE BIT ABOUT WHAT I DO AND WHAT MY LAB DOES JUST IN A VERY GENERAL WAY, UM, WHICH IS WE’RE INTERESTED IN CELL SURFACE ARE RECEPTOR AND HOW THEY ENGAGE LIGANDS. UH-UH, THE PROCESS OF CLUSTERING AND [INDISCERNIBLE] THAT OCCURS AFTER ENGAGING LIGANDS IS A PRESIT FOR SIGNALING AND THE CHINGS THAT OCCUR TO RELAY A SIGNAL ACROSS A MEMBRANE. AND WE’RE PARTICULARLY INTERESTED IN PLEIOTROPIC RECEPTOR SYSTEMS. THOSE ARE IN WHICH A GIVEN ARE RECEPTOR ENGAGED MORE THAN ONE LIGANDS ON A VARIETY OF CELL TYPES AND THESE PRODUCE DIFFERENT SIGNALALS AND HAVE DIFFERENT FUNCTIONAL [INDISCERNIBLE] AND THIS PROPERTY CERTAINLY IS A PROPERTY CYTOKINE RECEPTORS WHICH MOST OF YOU KNOW THAT I’VE WORKED ON, BUT IT IS A PROPERTY OF MANY OTHER RECEPTOR SYSTEMS. THE WAY WE LOOK AT THESE PROPERTIES IN THE LAB IS REALLY EVERYTHING JUST STRUCTURE IN BIOPHYSICS BUT THAT’SLY INFORMED BY PROTEIN ENGINEERING AND WE GENERATE MOLECULES BY WHICH WE CAN PROMECHANISMS FUNCTION BUT ALSO GENERATE MOLECULES THAT COULD BE POTENTIAL THERAPEUTIC LEADS. THIS IS THE OVERALL WINS WE VIEW ALL THE DIFFERENT RECEPTOR SYSTEMS WE WORK ON IN THE LAB. NOW, TODAY, UH, YOU MAY HAVE RECOGNIZED THESE RECEPTORS HERE AS BEING CYTOKINE RECEPTORS, AND THAT’S TRUE, BUT TODAY I’M GOING TO TALK ABOUT TWO RELATIVELY NEW PROGRAMS IN THE LAB. THAT THE PRINCIPLES ARE REALLY APPLICABLE TO ALL THE KINDS OF SYSTEMS WE WORK ON, AND THIS IS AGAIN LOOKING AT THIS ISSUE OF RECEPTOR LIGAND PLEIOTROPIC AND USING LIGAND TO REWIRE PLEIOTROPIC SYSTEMS — THE SECOND PART OF MY SEMINAR I’M GOING TO BE TALKING ABOUT A MORE GENERAL PROBLEM OF DEORPHANIZING — THIS IS A PROBLEM GIVEN 50% IN OUR GENOME REREMAIN ORPHANS AND I’LL TELL YOU ABOUT TECHNIQUES. EVENTUALLY THESE TWO TOPICS CURL BACK AROUND ON EACH OTHER AT THE END. STARTING OFF DISCUSSING WNT, UM, SO WNT, I’M NOT GOING GIVE MUCH OF AN INTRODUCTION, I THINK MANY OF US KNOW IT’S A DEVELOPMENTALLY MORE FA GENERAL — — TISSUE REGENERATION. IT’S A LIQUIDATED MOLECULE THAT IS CRETED AND ENGAGES TWO RECEPTORS, ONE IS THE FRIZZLED TRANCE MEMBRANE, A 7 TM RECEPTOR, ENGAGES A SECOND RECEPTOR CALLED LRP 6. THIS BINDING EVENT OCCURS EXTRA CELLULARLY AND IT INITIATES SEVERAL DIFFERENT SIGNALING PATHWAYS. THIS IS KIND OF A GREAT EXAMPLE OF PLEIOTROPIC IN CHA WE WNT BETA CADDIE NAH SIGNALING, ALSO THERE’S CONTROVERSY OVER WNTs WHETHER THEY PRODUCE G PROTEIN SIGNALING AND ALSO THERE’S NON-CANOTICAL SIGNALING. THERE’S A RATHER FUZZ DEFINITION OF THE MEKS THROUGH THE NON-CANOTICAL PATHWAYS BUT THROUGH THE CAOTICAL PATHWAYS IT’S UNDERSTAND. WNT ALSO HAS A VARIETY OF OTHER RECEPTORS SUCH AS RICK AND ROAR WHICH ARE THOUGHT TO SOMEHOW PARTICIPATE IN THE SIGNALING COMPLEX AS PART OF THE NON-CANOTICAL CASCADE. THE WAY I CAME INTO THIS PROBLEM WAS THIS ISSUE THAT THIS VERY INTERESTING BIOLOGICAL PROPERTIES OF WNT IN THAT THROUGHOUT DEVELOPMENT AND ADULTHOOD, WNTs HAVE A HOMEOSTATIC RANGE OF EXPRESSION AND WNT EXPRESSION PEAKS AND TROUGHS AT VARIOUS STATIONS. THESE PEAKS AND TROUGHS CAN BE NORMAL AND COINCIDE WITH TISSUE DEVELOPMENT AND STEM CELL DIFFERENTIATION AND SELF-RENEWAL. IT CAN ALSO BE THE RESULT OF INJURY. WNTs HELY INVOLVED IN TISSUE REGENERATION. CANCER CERTAINLY IS A CONSEQUENCE IN MANY CASES OF WNT SIGNALING DISREGULATION, AND TROUGHS, NEURAL DEGENERATION APPEARS TO HAVE SOME LINKAGE TO THE ABSENCE OF WNT. AND SO, THERE’S BEEN A LOT OF THERAPEUTIC INTERESTS IN HARNESSING THIS PATHWAY BUT WE REALLY HAVEN’T HAD ANY PICTURE OF HOW WNTs ENGAGE THEIR RECEPTORS. WHERE WE CAME INTO THIS ABOUT SIX OR SEVEN YEARS AGO WAS REALLY TRYING TO GAIN STRUCTURAL ACCESS THROUGH THE WNT SYSTEM AND THAT’S HOW WE LOOK AT STRUCTURE IN MY LAB. WE’VE THE COURTROOMED CRYSTAL STRUCTURES TO GAIN ACCESS SO WE CAN DO LIGAND ENGINEERING TO STUDY THESE PATHWAYS AND POTENTIALLY DEVELOP THERAPEUTICS. NOW THE OTHER ASPECT OF WNT THAT WAS SO COMPELLING FROM AN EXTRA CELLAR STANDPOINT WAS THAT WE HAVE 19 WNTs IN OUR GENOME AND TEN FRIZZLED. THERE’S NO KNOWN PARTICULAR LIGAND MATCHING CODE. WE DON’T KNOW WHY THERE ARE SO MANY OR WHETHER SPECIFIC WNTs ENGAGE SPECIFIC FRIZZ ARE L. WE THOUGHT THINKS A STRUCTURE OF WNT MIGHT HELP US UNDERSTAND THIS AND PERHAPS ENGINEER WNTs WITH PARTICULAR FRIZZLED SPECIFICITY THAT COULD BE USED TO INTERROGATE THESE PATHWAYS. SO WHY DID WE — IT TOOK US A LONG TIME AND AFTER ABOUT SIX YEARS OF EFFORT, WE SUCCEEDED BUT THE CHALLENGES WE FACE GOING INTO THIS WERE SOME UNUSUAL CHALLENGE FOR DETERMINING A STRUCTURE OF A PROTEIN. WNTs ARE LIPID MODIFIED AND THAT’S UNUSUAL FOR A SECRETED GROWTH FACTOR. THIS WAS REPORTED BY [INDISCERNIBLE] BACK IN 2003. WNTs ARE ALSO HEAVY GLYCOSYLATED, THEIR SIS TEEN RICH. THERE WAS NO KNOWN PREDICTIVE STRUCTURE OF WNTs. THE SEQUENCE REALLY PREDICTED THAT IT WAS GOING TO BE AN UNUSUAL STRUCTURE NO MATTER WHAT WE SAW. AND THIS PROBLEM HERE, THE INABILITY TO EXPRESS REKOFSH DANT WNTs HAS BROUGHT THE FIELD TO ITS KNEES. YOU CAN’T MAKE LARGE QUANTITIES OF THIS TO STUDY THEM. THERE ARE FEW VALIDATED WNT PAIRS WELCOMED GO AFTER SPECIFIC COMPLEXES. WE DO AN EXPRESSION SCREEN OF EVERY WNT FROM SEVERAL ORGANISMS AND WE SET ON ZINAPUS 8 AS ONE WE EXPRESSED LARGE QUANTITIES OF. WNTs. WE HAD A LARGE BODY OF FUNCTIONAL LITERATURE TO CORRELATE THE STRUCTURE. NOW, WE ALSO LOOKED AT THE WNT FRIZZLED SPECIFICITY HERE USING AN ALLY SA WHO WAS ORIGINALLY DEVELOPED BY MY NATHANS WHERE THE ALKALINE PHOSPHATES — — AND WE SCREENED THIS ACTIVITY WITH DIFFERENT FRIZZLED. FRIZZLE FIVE AND EIGHT LITTLE TO FRIZZLE FOUR AND SOME OF THOSE SOLUBLE FRIZZLE BINDING INHIBITORS. WE SET OUT TO FORM COMPLEXES BETWEEN ZENAFIS AND FRIZZLE 8 TO DETERMINE THE STRUCTURE. WNTs HAVE HISTORICALLY BEEN TREATED BY MEMBRANE [INDISCERNIBLE] THEY REQUIRE DETERGENT AND WE DEVELOPED A PURIFICATION FOR WNTs WHERE WE FUSED FRIZZLE 8 BINDING DOMAIN TO FC AND THEN CAPTURED WNTs AND THEN PURER FIED IT BY CLEAVING THAT OFF ON INFINITY COLUMN. THE PRISON LED AND THE WNTs, THEY DON’T CLEANLY [INDISCERNIBLE] THE WNTs AN FRIZZLE SEEM TO SLIDE PAST EACH OTHER A LITTLE BIT. MY POST DOC CLAUDIA, AFTER SEVERAL YEARS OF DEALING WITH THIS ISSUE, FINALLY DID A VERY SIMPLE EXPERIMENT WHERE SHE TRIED TO DO THE SAME THING AND SHE LEFT OUT THE DETERGENT. WHEN SHE DID THAT, YOU CAN SEE EVERYTHING SNAPPED INTO A ONE TO ONE COMPLEX AND ELUDED VERY NICELY ON GEL FILL STATION. THAT TOLD US THAT LIPID IS PROBABLY INVOLVED IN SOME WAY IN ENGAGING ITS ARE RECEPTOR BECAUSE WE COULD WITHDRAW DETERGENT AND THE WNT REMAINED SOLUBLE AND FORMED A TIGHT COMPLEX. THIS TO THE CRYSTALLIZATION AND STRUCTURE DETERMINATION WHICH I’M NOT GOING GO HEF ELY INTO BUT I JUST WANTED TO SHOW YOU THE STRUCTURE THAT FRANCIS WAS ELUDING TO EARLIER. WNT IS IN PINK HERE, AND THE FRIZZLED IS IN BLUE AND UH YOU CAN SEE THE THUMB AND THE INDEX FINGERER KIND OF GRASPING THE FRIZZLED CRD AND THAT’S THE LIPID GROUP THAT’S IMNATING. IT’S A POSTTRANSLATIONAL MODIFICATION THAT’S COMING OFF THE TIP OF THE WNT HERE AND IT’S INSERTING ITSELF INTO A DEEP GROOVE OF THE FRIZZLED CRD. NOW THAT DEEP GROOVE IS REALLY A — SORRY. YOU CAN SEE HOW DEEPLY THIS LIPID IS BURIED IN THIS GROOVE HERE, AND IT’S REALLY THE ONLY RECEPTOR LIGAND INTERACTIONS THAT I KNOW OF IN NATURE WHERE IT’S MEED GREATED IN PART BY POST TRANSLATIONALLY ADDED LIPID MODIFICATION. SO THIS ALSO RESOLVED WHAT IS THE ROLE OF THE LIPID? WHY ARE WITH WITH WNTs LIP DATED? TO ENGAGE THEIR RECEPTOR. IT USED TO BE THINKING WNTs WERE LIP DATED TO ANCHOR THEM TO THE PLASMA MEMBRANE AND PREVENT THEM FROM SECRETING TOO FAR AWAY FROM THE SITE THEY WERE EXPRESSED BUT CHEERILY IT’S INVOLVED IN INTERACTING WITH THE RECEPTORS HERE. NOW, ARE RECENTLY THE STRUCTURE OF THE SMOOTHED LIGAND BINDING DOMAIN WAS REPORTED AND SMOOTH IS CLASSIC RECEPTOR LIKE FRIZZLED, BUT IT’S THOUGHT AND MOVEMENT DOESN’T HAVE A LIGAND. WE CAN SEE THIS GROOVE ON THE FRIZZLED CRB IS ALSO PRESENT IN THE SMOOTH AND CRD, AND SO THERE’S NOW INCREASING EVIDENCE TO SHOW THAT SMOOTH AND PROBABLY DOES FIND SOME SORT OF A LIPID LIGAND LIKE FRIZZLED AND THE PRESENCE OF THIS GROOVE IN THAT STRUCTURE EVEN THOUGH WE DON’T HAVE A COMPLEX WITH SOMETHING, CERTAINLY LENDS CREDENCE TO THAT IDEA. UH NOW THE SECOND PART OF THE BINDING INTERFACE HERE OF WNT WITH FRIZZLED IS WHAT WE CALL THE INDEX FINGER AND THIS FINGER HERE IS JUST A LITTLE SHORT LOOP THAT’S PROTRUTDING INTO A CLEFT ON THE FRIZZLED CRD AND THE NOTABLE THING ABOUT THIS SITE RIGHT HERE IS THAT THE RESIDUES ON FRIZZLED THAT WNTs IS ENGAGING ARE MUCH MORE POLYMORPHIC THAN THE LIPID ENGAGING RESIDUES. SO THE RESIDUES ON FRIZZLED THAT THE LIPID BINDS TO ARE ALMOST COMPLETELY CONSERVED ACROSS ALL FRIZZLED, SO THAT’S PROBABLY NOT A SITE FOR SPECIFICITY DETERMINATION AND IT’S LIKELY THAT SPECIFICITY IS GOING TO BE MORE PART OF THIS INTERACTION SITE. WHAT I’VE DONE HERE IS I’VE LINED UP ALL THE FRIZZ ARE LED SEQUENCES AND COLORED IN BLUE THE RESIDUES THAT INTERACT WITH WNT AND YOU CAN SEE QUICKLY MANY OF THESE RESIDUES ARE DIFFERENT. SO THERE APPEARS TO BE FRIZZLED SUBTYPE SPECIFICITY ENCODED IN THIS SITE HERE. NOW, BEFORE MOVING ON TO THE ENGINEERING WORK WE’VE BEEN DOING, THIS IS THE WEIRDEST LOOKING PROTEIN STRUCTURE I’VE EVER SEEN, AND SO IT WAS VERY SATISFYING TO SEE THAT IN A WAY BUT IT REALLY RAISED THE QUESTION OF HOW DID THIS PROTEIN EVOLVE? HOW DID THIS FOLD EVOLVE? WE WORKED WITH BIZON ON THIS PROBLEM TO SEE IF WE COULD RECOGNIZE SUB DOMAINS THAT MIGHT HAVE BEEN STOLEN FROM OTHER PROTEINS. IT TURNS OUT TO BE A VERY INTERESTING THING. SO IF WE LOOK AT THE END TERMINAL DOMAIN OF WNT, THIS HAS THE LIPID ATTACHED TO IT DOWN HERE. IT’S A SAP SIN DOMAIN IT TURNS OUT. SAP SINS ARE LIPID BINDING PROTEINS THAT DELIVER LIPIDS TO OTHER PROTEINS. THE HUMAN SAP SINS DELIVER LIPIDS TO CB 1. AND SO IT APPEARS THAT SOME HOW WNT HAS BEEN HAS USED THAT DOE BUT COVALENTLY GROWN THIS LIPID ON THIS SAP SIN DOMAIN. THE SECOND WNT HERE THE IF LOOKED AT IN ISOLATION, IT LOOKS LIKE SIS TEEN NON-GROWTH FACTORS. IT’S A VERY LARGE CLASS OF CYTOKINES BUT IT’S ONLY HALF. NORMALLY THESE ARE DIMERS. SO WHAT WNT HAS DONE, IT’S FUSED TOGETHER HALF OF THE SIS TEEN NON-GROWTH FACTOR WITH THE SAP SIN DOMAIN TO CREATE THIS MOLECULE WITH TWO BINDING SITES AND TWO FUNCTIONALITIES. WHEN WE LOOK AT THE WAY HE’S SIS TEEN GROWTH FACTORS ENGAGE, THEY THE FINGERTIP JUST LIKE WNT ENGAGES RECEPTORS. IF WE CAN PROPOSE A EVOLUTIONARY PATH FOR HOW WNTs EVOLVED — PURELY HYPOTHETICAL — WE THINK MAYBE THE SAP SIN DOMAIN USED TO ABSTRACT LIPIDS FROM THE MEMBRANE AND DELIVER THEM TO THE WNT TO THE FRIZZLED LIE BEGAN BINDING DOMAIN WHILE THAT SIS TEEN NON-GROWTH FACTOR ENGAGED LRP. OVER TIME THIS LIPID GROUP BECAME COVALENTLY ATTACHED TO THE SAP SIN DOMAIN SO WHEN IT BOUND AND FRIZZLED IT DIDN’T LET GO. THERE MIGHT HAVE BEEN SOME GENE FUSION EVENT THAT ENGAGED LRP AND THAT RESULTED IN A BIFUNCTIONAL MOLECULE THAT ON ONE HAND IS ABLE TO ENGAGE FRIZZLED AND IN THE OTHER SIDE BRING IN LRP. SO THAT’S KIND OF THE WAY WE’RE THINKING ABOUT THIS AT THE MOMENT. SO NOW THE STRUCTURAL ACCESS, THE WNT AND SEEING HOW IT BINDS FRIZZLED WE CAN RE-ADD DRESS THIS QUESTION OF WHEN FRIZZLED SPECIFICITY. THE WAY WE’RE DOING THAT IS TO TRY AND ENGINEER FRIZZLED-SPECIFIC VARIANTS OF WNTs WE COULD USE AS EITHER ANTAGONIST OR AGONIST. THERE ARE SEVERAL PROBLEMS THAT WE’VE FACED IN TAKING THIS ON. ONE IS THIS RECURRENT ISSUE THAT WNTS ARE NOT WATER SOLUBLE. SO IT’S NOT LIKE A TYPICAL GROWTH FACTOR LIKE IL 2 WHERE YOU CAN MAKE LARGE QUANTITIES AND IT’S VERY WELL BEHAVED. THIS IS A PROBLEM THAT WE HAD TO FACE. THE SECOND ONE IS — AFTER CAN WE MAKE IT — THE SECOND IS CAN WE TAKE AGONISTS AND ANTAGONISTS THAT ARE FRIZZLE-SPECIFIC AND CAN WE BUILD A WNT MOLECULE THAT DOESN’T EVEN LOOK LIKE WNT. MAYBE WE COULD MIMIC WNT ACTIVITY IN A DIFFERENT STRUCTURAL SCAFFOLD. THAT’S WHAT I’M GOING TO TELL YOU ABOUT HERE. WE DID AN EXPERIMENT IN AN EFFORT TO MAKE A WATER SOLUBLE WNT BY MUTATING THE LIPID BINDING SITES ON WNT AND THEN EXPRESSING IT ON YEAST, AND THENAR ROR PRONE REACTION OF THIS GENERAL AND CREATING A LARGE LIBRARY ON YEAST AND SELECTING THEM AGAINST THE FRIZZLED LIGAND BINDING DOMAIN. THE LOGIC HERE WAS THAT MAYBE WE COULD ACQUIRE COMPENSATORY MUTATIONS OR A NON-LIPID DATED WNT THAT WOULD ALLOW EXPRESSION UNFOLDING ON THE SURFACE OF WNT. WE GOT WHAT LOOKED TO BE BINDERS TO FRIZZLED 8 AND 5 AND A LITTLE TO FRIZZLED 4. WE WERE ENCOURAGED MAYBE WE HAD THIS MOLECULE. WHEN WE SEQUENCED THE GENE WHAT WE FOUND WAS THAT EVERYTHING FROM WNT HAD BEEN DELETED OUT EXCEPT THE TIP OF THIS FINGER RIGHT HERE. THAT WAS REMEANING ON THE YEAST AND IT WAS BINDING TO THE CRD. THAT FRINGER TIP REKA P PITCH LATES THE SPECIFICITY OF THE FULL-LENGTH WNT THAT I SHOWED YOU EARLIER. THAT REALLY TOLD US MOST OF THE SPECIFICITY OF WNT IS IN THIS SITE RIGHT HERE. SO WE DID A FEW EXPERIMENTS TO LOOK AT THIS CAREFULLY WHERE WE FIRST WE CUT OUT THE FINGERTIP OF ALL THESE DIFFERENT WNTs AND WE JUST MEASURED THEIR BINDING ON TO THESE DIFFERENT FRIZZLES. WE FOUND UNEXPECTED RESULT WHICH IS THAT PRETTY MUCH ALL OF THE WNTs LIKE FRIZZLED 5 AND 8 AND HAVE VERY LOW AFFINITY OR NO AFFINITY TO THE OTHER FRIZZLES. SO WE WERE EXPECTING TO SEE A MORE POPULATED MATRIX HERE. THEN WE DID A LITTLE MORE HARD NOSED EXPERIMENT WHERE INSTEAD OF LOOKING AT THE FRINGER TIP, WE GRAFTED THE FINGERTIP FROM EACH ONE OF THESE WNTs BACK INTO FULL-LENGTH ZENAPUS AND ASSESSED THE BINDING OF THE FULL-LENGTH WNT NOW. YOU CAN SEE IT RECAPITULATED THE SAME SPECIFICITY AS THE FINGERTIP. AND SO THIS IS PUZZLING BECAUSE WHY DO WE HAVE ALL THESE OTHER FRIZZLES IF THEY’RE ONLY BINDING TO THREE OR — TWO OR THREE OUT OF ALL OF THEM? THIS ALSO IS CONSISTENT BUT IT ALSO CONTRASTS A BIT WITH A PAPER FROM JEREMY NATHANS WHERE HE LOOKED AT THE FUNCTIONAL, HE DID AN INTERACT TOME OF 16 WNTs AGAINST THE 10 FRIZZLE LS USING THE SIGNALING REPORTER ASSAY, KIND OF AN INTERACT TOME EXPERIMENT. HE FOUND FRIZZLES FIVE AND EIGHT ARE DEFINITELY THE MOST PREFERRED ONE BUT THERE ARE THE POCKETS OF WEAK ACTIVITY WITH THESE OTHER WNTs AND FRIZZLES THERE. SO IF THERE IS A RECEPTOR/LIE GRAND MATCHING CODE HERE, IT’S VERY BLURRED, AND TO REALLY ASK THIS QUESTION CLEANLY, WE WANTED TO DEBLUR THIS TYPE OF MATRIX HERE AND MAKE IT LOOK SOMETHING LIKE THIS WHERE WE WOULD HAVE WNTs THAT SPECIFICALLY ENGAGED ONE OR SPECIFIC COMBINATION OF FRIZZLE SO WE COULD THEN GO INTO BIOLOGICAL ASSAYS AND ASK QUESTIONS. AND WE’RE IN THE PROCESS OF DOING THIS NOW AND THE WAY WE’VE DONE THIS SINCE YOU CANNOT REALLY WORK WITH A FULL LENGTH WNT MOLECULE BECAUSE OF THE BIOCHEMICAL PROBLEMS ASSOCIATED WITH THE LIPID SO WE’RE DISPLAYING THE FINGERTIP ON YEAST AND WE’RE CREATING THIS PSYCH LA PEPTIDE LIBRARIES. WE’RE SELECTING THEM ON SPECIFIC FRIZZLES. HERE’S A SELECTION OF A LIBRARY AGAINST FRIZZLE LED EIGHT. YOU CAN SEE AS WE SELECTED ENRICH, WE GROW THIS POPULATION OF ENRICHED FINGERS THAT WE THEN GRAFT BACK INTO FULL-LENGTH WNT AND ASK ABOUT ITS SPECIFICITY. AND DOING THAT, WE HAVE BEEN ABLE TO CREATE MONO-SPECIFIC AND BI-SPECIFIC VERSIONS OF ZENAFIS ONE EIGHT. WE’RE IN THE PROCESS OF TESTING THESE WITH RANDY MOON AND SEVERAL OTHERS TO ASK WHETHER THE DIMINISHED SPECIFICITY OR ALTERED SPECIFICITY, WHAT ARE THE CONSEQUENCES IT HAS? WE’RE IN THE PROCESS OF DOING THIS, BUT THIS APPROACH APPEARS TO WORK IN PRINCIPLE. THE SECOND APPROACH THAT WE’RE USING IS TO CREATE WNTS THAT DON’T LOOK LIKE WNTs. IN OTHER WORDS, WNT SURROGATES AND THIS IS ALSO SOMETHING WE’VE DONE A LOT OF WITH CYTOKINES AS WELL. OUR APPROACH HERE WAS — SO WE ENGAGED DAVID BAKER WHO’S A PROTEIN DESIGNER EXTRAORDINARY AS UNIVERSITY OF WASHINGTON MEAN DAVID, YOU CAN GIVE HIM ONE OF THESE WALLS LECTURES, I’M NOT SURE. WHAT WE PRESENTED HIM WITH WAS THE STRUCK UH CHUR OF WNTS WITH THAT LIPID IN THIS GROOVE OF THE FRIZZLED CRD HERE, AND CHALLENGED HIM TO COME UP WITH A PROTEIN THAT WOULD FIT INTO THE GROOVE — REPLACE THE LIPID WITH LET’S SAY AN ALPHA HELIX, BUT THEN READ OUT THE PEAKS OF THESE PRESSED ON THE SIDE OF THIS TROUGH RIGHT HERE WHERE RESIDUE-SPECIFIC FRIZZLED RESIDUES THAT ARE SPECIFIC TO EACH SUBTYPE EXISTS. SO ONE MIGHT HAVE A LIPID MIMIC THAT IS FRIZZLED-SPECIFIC. AND THIS IS THE PROTEIN DESIGN THAT HE CAME — THAT HE AND LUKE DANGER IN HIS LAB CAME UP WITH. IT’S A FOUR HELIX BUNDLE PROTEIN WHERE ONE OF THESE HE LA SEES WOULD SPAN THE LINTH OF THE GROOVE HERE AND THESE OTHER HE LA IS IS WOULD IN MANY WAYS IF YOU’RE INTO MHC MOLECULES THAT WOULD BRACKET THE SIDES OF THE GROOVE HERE MUCH IN THE WAY A T CELL RECEPTOR SEES A PEPTIDE MHC. THIS WAS THE DESIGN AND WE SPENT A FAIR AMOUNT OF TIME WITH DAVID AND LUKE ENGINEERING THE PROTEIN AND WE FINALLY CAME UP WITH THESE VARIANTS THAT ONE OF THEM CALLED B 12 IN WHICH YOU CAN SEE HERE IS THAT B 12 BINDS TO FRIZZLED FIVE AND EIGHT LIGAND BINDING DOMAIN WITH SINGLE DIGIT OR AFFINITY AND IT DOESN’T BIND TO ANY OTHER. ONE REASON WHY THIS IS SIGNIFICANT IS BECAUSE MAKING FRIZZLE-SPECIFIC ANTIBODIES HAS NOT BEEN ACCOMPLISHED. SEVERAL DRUG COMPANIES WANT TO TARGET FRIZZLED AS AN ANTICANCER TARGET TO BLOCK WNTs ACTIVITY HAVE GENERATED ANTIBODIES BUT THEY ALL CROSS REACTION TO FRIZZLED SUBTYPES. SO WE’D LIKE TO MAKE SUBTYPE VAERNTS AND IT APPEARS WE’VE DONE THAT IN THIS DESIGN HERE. IT ALSO HAS WEAK BLOCKING ACTIVITY. THIS IS A CARCINOMA CELL LINE THAT’S WNT SENSITIVE. YOU CAN SEE THAT WE CAN COMPETE WITH TWO DIFFERENT DESIGNS FOR WNT ACTIVATION OF BETA CATIMIM IN A DOSE-DEPENDENT FASHION BUT IT’S A WEAK INHIBITION. WHY IS THAT? SO THIS SLIDE IS KIND OF AN INTERESTING SLIDE. WHAT I’M SHOWING YOU HERE IS THE DESIGN FROM DAVID OF WHAT THE PROTEIN SHOULD LOOK LIKE BINDING TO THE FRIZZLED CRD. WHAT YOU’RE ABOUT TO SEE IS THE DESIGN IS GOING TO DISAPPEAR AND WE SOLVED THE CRYSTAL STRUCTURE OF WHERE IT ACTUALLY BINDS. SO, UH — THAT’S THE DESIGN, THERE’S WHERE IT ACTUALLY BINDS. SO, WHEN YOU LOOK AT THAT, YOU COULD SAY, ALL RIGHT, WELL, THEY DIDN’T QUITE GET IT, THINK BLEW IT, BUT THEY GOT CLOSE, AND YOU CAN SEE THAT THE REASON WHY THIS IS PROBABLE HI PARTIALLY INHIBITORY IS THE HELIX ONLY INTERRUPTS A SMALL PART OF THIS LIPID BINDING GROUP HERE. SO IT’S WEAKLY INHIBITORY, BUT THE SURPRISING THING ABOUT THIS STRUCTURE IS THAT THESE HELIXES SIT DOWN ON FRIZZLED IN THE PERFECT SPOT TO READ OUT SPECIFICITY. IN OTHER WORDS, THIS TRANSCRIPT FEIGN RESIDUE THAT’S STICKS UP BETWEEN THESE TWO ALPHA HELIXES IS SUB B TYPE SPECIFIC. IN FACT WHEN WE LOOKED AT OTHER RESIDUES THAT THESE HELIXES ARE CONTACTING, YOU CAN SEE HOW MANY OF THEM ARE SUBTYPE-SPECIFIC. SO EVEN THOUGH WE MAY HAVE MISSED IN CREATING THE PERFECT WNT INHIBITOR, WE MAY HAVE HIT UPON A GREAT SOLUTION FOR A SCAFFOLD THAT CAN READ OUT FRIZZLED SPECIFICITY IN THAT WE MIGHT BE ABLE TO MAKE FRIZZLED-SPECIFIC ANTAGONISTS. RECENTLY WE’VE MADE USE OF THE SCAFFOLD FOR ANOTHER REASON THAT IS REALLY GOING BE A BIG DIRECTION FOR WHERE WE’RE GOING WITH THIS PROJECT. SO THE WAY CANODICAL WNT SIGNALING WORK IS IT ENGAGES FRIZZLED AND THEN RECRUITS LRP TO FORM A SIGNALING COMPLEX. MANY CELL SURFACE RECEPTORS SIGNAL JUST BY DIMERIZATION. IT APPEARS THAT NO ONE HAS REALLY KNOWN WHETHER WNT SIGNALS THROUGH SIMPLY BRINGING LRP OR WHETHER SOME SPECIFIC CONFORMATIONAL CHANGES ARE REQUIRED THROUGH FRIZZLED THAT WOULD BE REQUIRED FOR SIGNALING. SO NOW WE HAVE A SURROGATE PROTEIN THAT BINDS TO THE FRIZZ ARE LED CRD, IT DOES NOT INDUCE SIGNALLING IN FRIZZLED, BUT WHAT IF WE USE THIS TO LINKER TO A NATURAL PROTEIN THAT BINDS TO LRP AND ENFORCE PROXIMITY TO LR P FOSHGS FRIZZLED; WILL THIS SIGNAL? WE RECENTLY DID THIS EXPERIMENT AND THE ANSWER IS, IT DOES. SO HERE’S A BETA CATINAN ASSAY AND HERE’S ZENAPUS ONE EIGHT IN A DOSE GENT ACTION — — AND IT DOES SO IN A FRIZZLED-SPECIFIC WAY. IT DOES NOT ACTIVATE FRIZZLE TWO CONTAINING CELLS, IT ACTIVATES FRIZZLED EIGHT AND FIVE-CONTAINING CELLS. NOW WE HAVE A WATER SOLUBLE SURROGATE ACTIVIST OF WNT. WE VERY RECENTLY GOT DATA FROM RANDY MOON WHERE THEY HAVE BEEN LOOKING AT OUR SURROGATE AGONISTS IN THIS SPHERE GROWTH AIS I WHICH THEY EXTRACT STEM CELL FROM A MOUSE FRAME AND THESE CELLS ARE SELF-RENEWAL AND IS CONTROLLED BY WNT AND THEY’VE BEEN USING WNT 3 A TO DO THIS. YOU CAN SEE HOW THESE NEUROSPHERES EXPAND IN SIZE OVER TIME. WHEN YOU LOOK AT OUR SURROGATE, IT’S MUCH BETTER THAN EITHER OF THE WILD TYPE WNTs HERE. WE’RE VERY EXCITED ABOUT THIS, AND YOU KNOW AS PROJECTS TEND TO DO IN SCIENCE, THEY KIND OF MEANDER ALONG IN DIFFERENT WAYS AND YOU KEEP DOING THINGS AND EVENTUALLY YOU SEE SOMETHING YOU DIDN’T EXPECT AND NOW YOU’RE GOING OFF IN THAT DIRECTION. NOW WE HAVE A WATER SOLUBLE WNT AGONIST THAT’S EASY HI PRODUCED IN VERY LARGE QUANTITIES THAT WE THINK AND ARE EXPLORING MIGHT FUNCTIONALLY FEE KNOW COPY WNT IN EVERY WAY. SO THIS WOULD BE REALLY BE A BOOM TO THE FIELD AND IT’S ALSO ENGINEERABLE. SO THAT’S REALLY WHERE WE ARE AT THE MOMENT WITH THE WNT ENGINEERING, AND HOPEFULLY WITH MAKING A WHOLE BATTERY OF THESE FRIZZLE-SPECIFIC AGONISTS AND ANTAGONISTS WE’D BE ABLE TO REVISIT THESE KINDS OF QUESTIONS THAT THE ROLE ARE OF WNT FRIZZLE SPECIFICITY AND THESE PROCESSES WITH AN ENTIRELY NEW APPROACH. OKAY. SO I’M GOING SEGUE HERE IN A RATHER JARRING WAY THAT IT MAY SEEM RIGHT NOW BUT IN THE END YOU’LL SEE THERE’S SYNERGY WITH WHAT I JUST SPOKE ABOUT. GOING INTO THIS TALKING ABOUT DEORPHANIZING ARE RECEPTORS. AND SO THE REASON WHY I HAVE THIS SLIDE, I P PUT THIS WATER COLOR FROM THE 36 VIEWS OF MOUNT FUJI, I WAS THINKING STRUCTURAL BIOLOGY IS ESSENTIALLY VIEWING A MOLECULE OR PROBLEMS FROM DIFFERENT VANTAGE POINTS, AND SO, YOU KNOW, THAT’S WHAT WE DO. IT’S A DESCRIPTIVE FIELD AND SO WE’VE HAD ONE VIEW OF MOUNT FUJE HERE HERE AND THIS IS THE SECOND VIEW OF MOUNT FUJI HERE. IF THAT HELPS TO MAKE THE TRANSITION FOR YOU, FEEL FREE TO USE THAT. ALL RIGHT. SO I THINK WE CAN ALL AGREE THAT CELL SURFACE RECEPTORS AND THEIR LIGANDS ARE IMPORTANT. YET MOST OF THEM ARE ORPHANS, AND THE NUMBER OF ORPHANS, THE PERCENT OF ORPHANS ARE GENOME ENCODES TWO OR THREE THOUSAND CELL SURFACE RECEPTORS, APPROXIMATELY EQUAL NUMBER OF SECRETED PROTEINS THAT WOULD PAIR WITH THEM. THE ESTIMATES ARE IN THE RANGE OF 50% OF CELL SURFACE ARE ORPHANS AND 50% OF SECRETED LIGANDS DO NOT HAVE KNOWN RECEPTORS. THIS HAS BOTHERED ME OVER THE YEARS BECAUSE AS A STRUCTURAL BIOLOGISTS ONE OF THE FRUSTRATIONS OF OUR FIELD IS THAT WE’RE ALWAYS IN THE POSITION OF DESCRIBING DISCOVERIES THAT OTHERS MAKE. AND SO WE COME IN USUALLY LATE LIKE IN THE WNTs FIELD, WE CAME IN LATE AFTER ALL THE FUN BIOLOGY HAS BEEN WORKED OUT, AND SO WHAT I REALLY WANT TO DO IS BE AHEAD OF THAT CURVE AND DISCOVER NEW INTERACTIONS AND THEN FULLY CHARACTERIZE THEIR FUNCTIONS AS WELL AS THEIR STRUCTURES, AND SO THIS PROBLEM OF CELL SURFACE RECEPTOR DEORPHANNIZATION SEEMED LIKE STHENG THAT AS A BIOCHEMIST THAT LAB MIGHT BE ABLE TO HAVE A NEW APPROACH TO DO THAT TO HAVE A USE IN THIS REGARD. RECEPTORS COME IN MANY DIFFERENT TYPES. THEY BIND TO SOLUBLE MEDIATORS. THEY BIND TO TRANS AND CYSTS. WE FOCUSED ON THESE RECEPTORS THAT MEDIATE CELL JUNCTIONS, AND MANY OF THEM ARE ARE IG SUPER FAMILY CLASS RECEPTORS AND LIEU SEEN RICH REPEAT ARE RECEPTORS. IG SUPER FAMILIES ARE THE LARGEST FAMILY OF CELL SURFACE RECEPTORS WE HAVE IN OUR GENOME AND THAT OTHER ORGANISMS HAVE IN THEIR GENOMES AND LUCY RICH REPEAT PROTEINS ARE SECOND AND MOST OF THEM ARE AS WELL ABOUT ORPHANS. DEORPHANIZING ARE RECEPTOR IS TECHNICALLY CHALLENGING BECAUSE THEY’RE ON MEMBRANES, MODIFIES, GLYCOSYLATED. SYSTEMS BIOLOGY TYPE OF PROTEOMICS TYPE OF TAP TAGGING APPROACHES ARE VERY MESSY, THEY DON’T WORK WELL FOR CELL RECEPTORS. SO WHAT WE DO IN MY LAB IS WE’VE SPENT A LONG TIME LOOKING AT THE EXTRA CELLAR DOMAINS T — OF RECEPTORS. WE THOUGHT, WHAT IF WE EXPRESS EVERY EXTRA CELLAR DOMAIN OF THE GIVEN FAMILY OF RECEPTORS AND ARRAY THEM IN A PARALYZED WAY LOOKING FOR INTERACTIONS; WHAT WOULD WE FIND? COULD WE FIND BIOCHEMICALLY BONA FIDE HITS AND THEN WORK BACKWARDS TO THE FUNCTION? THAT’S THE PROJECT I’M GOING TO TELL YOU ABOUT TODAY. WE WENT INTO JESOFALA BECAUSE IT HAS FEWER GENES AND WE CATALOGED RECEPTORS, ABOUT 202 OF THEM. WE EXPRESSED ALL OF THEIR EXTRA CELLAR DOMAINS. TO DO THIS PEAR-WISE INTERACTION WHICH COMPRISED ABOUT 40,000 EXPERIMENTS LOOKING AT 20,000 UNIQUE PEAR-WISE INTERACTIONS. THE WAY WE DID THIS WAS A RELATIVELY SIMPLE ASSAY WHERE WE EXPRESSED EACH EXTRA CELLAR DOMAIN IN THE BAKED FORM AND THE PREY FORM. THE BAIT FORM WAS CONNECTED TO AN FC AND THE PREY FORM WAS ALKALINE PHOSPHATASE AND IT WAS PENTMORERISED. MANY ADHESION RECEPTORS HAVE VERY LOW AFFINITY. WE USED PENTMORERYIZATION TO CAPTURE THESE INTERACTIONS. WHAT WE DID IS WE EXPRESSED IN CELLS THE ECDs OF ALL 202 IN THESE IN BOTH BAIT AND PREY, WE ARRAYED THEM IN BLOCKS ALL BY HAND, AND THE EXPERIMENT LOOKS LIKE THIS. YOU HAVE YOUR PROTEIN A, YOU CAPTURE YOUR BAIT, FC, YOU COME ALONG WITH YOUR PREY, AND IF IT BINDS, IT TURNS BLUE. AND THAT’S WHAT THE ASSAY LOOKS LIKE. YOU CAN STATISTICALLY ANALYZE THE SIGNAL AND NOISE ALL DAY LONG IN THAT PLATE, BUT AT THE END OF THE DAY THE WAY WE MADE THE CALL IS WE HELD THEPLAY PLATE UP TO THE LIGHT AND SAID THERE’S A BLUE SPOT AND THAT’S 100% ACCURATE. OKAY. THAT’S WHAT THE MATRIX LOOKED LIKE AFTER EXP EXPERIMENT WAS COMPLETED. INTERNAL CONTROLS FOR KNOWN INTERACTIONS AND THIS WAS OUR YIELD. FOR HOMO FILLIC INTERACTIONS, WE RECOVERED 20 HITS OF WHICH 16 WERE PREVIOUSLY KNOWN AND FOUR WERE NOVEL. HETERO FILL LICK, WE SCREENED 20,000 POSSIBLE PAIRS, FOUND 86 INTERACTIONS OF WHICH 80 OF THEM WERE NOVEL. NONE OF THESE WERE IDENTIFIED FROM PROTEOMIC-TYPE OF CELL, YOU KNOW, MASSIVE IP-TYPE OF SYSTEMS BIOLOGY APPROACHES. I THINK ONE OF THE REASONS THAT OUR RESULTS WERE SO CLEAN IS THAT IT’S A PAIR-WISE APPROACH; ONE AGAINST ONE. THIS IS A SUMMARY OF THE NODES WE FOUND HERE. WE FOUND — SO THE BLUE LINES INDICATE PREVIOUSLY UNKNOWN INTERACTIONS HERE. THIS IS THE LARGEST FAMILY WE FOUND. IT’S A FAMILY OF IG SUPER FAMILY PROTEINS CALLED THE DIPS AND THE DPRs. THESE WERE THESE FORMALLY GENE EXSESSION NUMBERS. WE FOUND A COMMON LIEU SEEN RICH REARE PEAT FAMILY WHICH SEEMED TO INTERACT WITH SOME OF BOTH SIDES OF THIS EQUATION. WE ALSO DEORPHANIZED MANY MEMBERS OF THIS OTHER ARE RECEPTOR/LIGAND FAMILY HERE. BEATEN PATH AND SIDESTEP. WE ALSO RECOVEREDED THESE KNOWN HITS HERE; THE ARE RED LINES INDICATE INTERACTIONS WIHAT WAS PREVIOUSLY KNOWN. THAT WAS SATISFYING TO SEE. THE NICE THNG IS WE FOUND FAMILIES. P SO THESE EVOLUTIONARILY, THESE PROTEINS ARE EVE LUGS NARLY LINKED AND THEY ENGAGE OTHER FAMILIES THAT ARE ALSO EVOLUTIONARILY. FOCUSING ON THIS DIP DPR NODE RIGHT HERE, KNOCKOUTS OF THE DPR HAVE BEEN KNOWN. THESE WERE CALLED DEFECTIVE P PROBOSTIC RESPONSE. THESE WERE KNOWN TO BE ACTIVE — — BUT THEIR LIGANDS WON’T KNOWN. NOW WE HAVE THEIR LIGANDS AND CLEARLY MOST OF THESE LOOK TO BE ACTIVE IN THE NERVOUS SYSTEM. WE VALIDATED THESE HITS BIO CHEMICALLY WHERE WE EXPRESSED RECOME BY DENT EXTRA CELLAR DOMAINS OF THESE PAIRS AND WE MEASURED BINDING AFFINITIES. ONE IMPORTANT VALIDATION CRITERIA SHEER THEY GIVE US MATHEMATICAL [INDISCERNIBLE] NON-SPECIFIC INTERACTIONS DO NOT EXHIBIT THAT KIND OF BEHAVIOR. THAT’S A VERY ROBUST TEST THAT THESE KIND OF HITS ARE PROBABLY BIOLOGICALLY RELEVANT. THEN SO AS ONE EXAMPLE, WE’RE FINDING AFFINITY FOR DPR SIX TO A DIP IS ABOUT ONE MICRO MOLAR AND THE AFFINITY OF THIS COMMON DIP FOR ONE OF THE CGs DOWN HERE IS ON THE ORDER OF THE SAME. THESE ARE LOW AFFINITY INTERACTIONS CONSISTENT WITH CELL SURFACE ADHESION MOLECULES. WE THINK THIS COMMON DIP MIGHT BE SOME SORT OF NEGATIVE REGULATOR OF THE INTERACTIONS OF THESE PAIRS. NOW WE’VE BEEN WORKING WITH KYLE AT CAL TECH WHO ASSESSED THE FUNCTIONS OF THESE INTERACTIONS AND WHAT KYLE HAS BEEN USING IS THE ACTUAL ALKALINE PHOSPHATES WE USED IN OUR EXPERIMENTS TO STAIN LIVE SECTIONS. IT’S AN ANTIBODY LABELING METHODOLOGY. JUST TO SHOW YOU SOME OF THE DATA, THIS IS THE DIP THAT SEEMS TO BIND TO MOST MEMBERS OF THAT FAMILY AND YOU CAN SEE THAT IT’S EXPRESSED PRETTY MUCH IN ALMOST ALL SUBSETS OF NEURONS HERE. WHEREAS, OTHERS — OH, WE ALSO DID A EXPERIMENT WHERE WE LOOKED AT A PARTICULAR PAIR WE FOUND BINDING TO D P PR 11 AND THE STAINING IS VERY NICE HERE IN A WILD TYPE ANIMAL, BUT IN THE CG 14 I’VE TO ONE KNOCKOUT, WE LOSE THE STAINING. WE’VE GONE ON UH NOW TO MAKE WHAT ARE CALLED MIMIC CLONES IN WHICH THE GENE IS NOT KNOCKED OUT, IT’S REPLACED BY GFP. THAT’S A MARKER THAT IT’S STILL THERE. WHAT YOU CAN SEE HERE IS A HETERO ZYGOTE M MIK MIK WHERE
THE GREEN CELLS REPRESENT THE KNOCKOUT AND THERE’S PARTIAL STAINING HERE, BUT IN THE HOMO ZYGOTE MIMIC YOU SEE THE NO STAINING — — WE KNOW IT APPEARS TO OCCUR IN LIVE CELL EMBRYOS. THIS IS SOME RECENT DATA FROM ARE KYLE AS WELL ON SOME OF THESE OTHER INTERACTING MEMBERS AND THE POINT IS THAT EACH ONE OF THESE DIFFERENT GENES AND THEIR PARTNER ARE ARE EXPRESSED IN DISTINCT SUBSETS OF NEURONS. EACH WITHIN OF THESE GREEN NODES IS A NEURON BUT THEY’RE EXPRESSED IN THE SAME PLACE. IT’S REASONABLE TO THINK THEY COULD HAVE AN INTERACTION IN A LIVE ANIMAL. NOW, OF COURSE I’M A STRUCTURAL BIOLOGISTS AND WE’RE INTERESTED IN THIS ADHESION FAMILY HERE UNDERSTANDING THE STRUCTURAL BASIS OF THE INTERACTION. SO WE RERECENTLY SOLVED THE CRYSTAL STRUCTURE OF ONE OF THESE NEW DEORPHANIZED PAIRS AND THAT’S SHOWING YOU RIGHT HERE THAT THESE ARE TWO IG DOMAINS. THIS IS A TWO IG DOMAIN OF THE CG, AND ONE IG DOMAIN OF THE DP ARE R SIX AND THEY FORM THIS ALMOST ORTHOGONAL TYPE OF INTERACTION HERE AND UNTIL WE HAVE NOR DATA ABOUT WHAT THESE ARE DOING IN THE FLY, WE CAN’T REALLY SAY MUCH MORE ABOUT THAT, BUT IT IS NOTABLE BECAUSE WE ALSO HAVE BEEN LOOKING AT STRUCTURES OF ANOTHER SET OF ADHESION FAMILY MEMBERS HERE, THE ROUGHEST STICKS AND STONES FAMILY ALSO CALLED NEPHRONS. THEY’RE CELL ADHESION MOLECULES THAT, UM, — WELL MY COMPUTER LOOKS FINE. THERE WE GO, OKAY. IT WAS A LITTLE LOOSE HERE. OKAY. OKAY. SO THESE FORM THE FILTRATION BARRIER IN THE KIDNEY SLIT DIAPHRAGM IN HUMANS. THEY ALSO HAVE OTHER FUNCTIONS IN IS I NAPT GENESIS, THEY FORM THE SYNAPSE — THEY ALSO FORM OTHER CELL/CELL JUNCTIONS. AND THESE MOLECULES ARE LONG IG SUPER FAMILY-CONTAINING RECEPTORS AND WE CRYSTALLIZED A COMPLEX BETWEEN CIG ONE AND TWO AND IT HAS THIS STAIN ORTHOGONAL TOPOLOGY HERE. WE ALSO CRYSTALLIZED THESE OTHER MEMBERS OF THIS FAMILY AND THEY ALL SHARED THE SAME ORTHOGONAL SOPOLOGY AND WHEN YOU COMPARE THAT TO THE DPRT NEW DEORPHANIZED DPR INTERACTION, THEY’RE ABSOLUTELY DEAD-ON. WE’RE THINKING MAYBE THESE ADHESION MOLECULES HAVE THIS SHARED ARCHITECTURE BECAUSE IN SOME WAY IT’S CONDUCIVE TO THE FORMATION OF A FUNCTIONING SYNAPSE. AND WHAT HAS BOLSTERED THIS IDEA MORE IS WE’VE LOOKED AT THE WHOLE LENGTH DOMAINS OF THESE MOLECULES AND YOU CAN SEE HOW THEY’RE QUITE RUNLD. WHEN THEY BIND, THEY FORM “L” SHAPE. THEY’RE FORMING THIS ROD PROPAGATED ALONG BOTH AXIS HERE. THAT “L” SHAPE WOULD BE REPRESENTED IN THIS SYNAPSE HERE WHERE WE REMODELLED IT HERE. THIS RAISES THE QUESTION: DOES THIS STRUCTURE MATTER TO THE SYNAPSE? IS THE RIGIDITY IMPORTANT AND IS THE DOCKING IMPORTANT? WE COLLABORATED WITH SHIN AND WE USED THIS SYNAPSE FORMATION ASSAY IN C EL GAND VOL VA. YOU CAN SEE CIG TWO LOCALIZES TO THIS SYNAPSE RIGHT HERE WHEN WILD TYPE CIG ONE IS PRESENT. WHEN YOU MUTATE IT THE CIG TWO DRIFTS AWAY AND YOU DON’T GET A PROPER SYNAPSE FORMATION THERE. WHAT WE DID WAS WE MADE MUTATIONS IN THIS INTERFACE RIGHT HERE AND ASKED ABOUT THE SYNAPSE FORMATION, AND WHAT YOU CAN SEE IS THAT THE FREE ENERGY OF THE MUTATIONS WE MADE IN THE SYNAPSE CRACK PERFECTLY. THIS MUTATION IN THAT INTERFACE ALMOST COMPLETELY BLOWS AWAY BINDING AND WE LOSE ALMOST ALL SYNAPSE FORMATION, WHEREAS MUTATIONS THAT ARE LESS OF AN EFFECT HAVE LESS. SO WHAT THIS MEANS IS THAT THE IN VIVO READ OUT IS A FAITHFUL REPRESENTATION OF THE FREE ENERGY OF THE INTERACTION IN THAT INTERFACE HERE. AND THAT’S A VERY UNUSUAL POSITION TO BE IN. SO WE WENT ON AND WE DID TWO THINGS. WE INSERTED FLEXIBLE LINKERS BETWEEN THE IG DOMAINS, SO CIG ONE AND CIG TWO, AND YOU CAN SEE HOW COMPARED TO THE WILD TYPE, INTRODUCING FLEXIBLE IMPAIRS THE ABILITY TO FORM SYNAPSES. THE SECOND THING WE DID WAS THAT WE REPLACED THE INTERACTING IG DOMAINS WITH IG DOMAINS FROM OTHER IMMUNO GLOW BU LIN THAT BINDS IN A DIFFERENT DOCUMENT [INDISCERNIBLE] WHEN WE DID THAT WE ALSO IMPAIRED SYNAPSE FORMATION. WITHIN THE “L” SHAPED APOLOGY AND THE RIGID DI OF THESE MOLECULES IS IMPORTANT FOR THE FUNCTIONAL FORMATION OF A WORKING SYNAPSE AND PERHAPS THIS IS TRUE ACROSS A LARGER BODY OF ADHESION RECEPTORS INCLUDING THAT WE JUST TALKED ABOUT PREVIOUSLY. SO THE WAY THIS MIGHT LOOK IS THAT WITHIN THE SYNAPSE YOU HAVE THIS “L” SHAPED STRUCTURE AND IT FORMS A TIGHTLY-PACKED RAY THAT IN THREE DIMENSIONS FORMS KIND OF A MESH WORK-LIKE STRUCTURE AND A PARTICULAR SHAPE AND PROPERTIES OF THIS COULD BE IMPORTANT FOR CLOSE-PACKING INDUCED MOLECULES. SO I THINK I’M GOING TO STOP AT THIS POINT, UM, AND THANK THE PEOPLE WHO PARTICIPATED IN THESE PROJECTS. CLAUDIA REALLY CARRIED THE BALL ON THE WNT PROJECT FOR PRETTY MUCH ALL OF THE STRUCTURAL WORK AND WE’RE HAVING GREAT COLLABORATIONS WITH DAVID BAKER AND RANDY MOON ON GETTING BACK INTO THE FUNCTIONAL ASPECT OF THE PROJECT. OSCON IS AN ASSISTANT PROFESSOR AT UNIVERSITY OF CHICAGO, REALLY DID ALL OF THE INTERACT TOME AND CIG BIO CHEMICAL WORK. AND WE COLLABORATED WITH SHIN ON THE SOME OF THE RECENT FUNCTIONAL WORK. AND [INDISCERNIBLE] WAS A HUGE COLLABORATOR FOR THE INTERACT TOME PROJECT. JUST BEFORE CLOSING UP HERE PERMANENTLY, I JUST WANTED TO TELL YOU THAT REALLY THIS IDEA OF PAIRING RECEPTORS WITH THEIR LIGANDS IN THIS KIND OF SINGULAR PAIR-WISED WAY IS A POWERFUL APPROACH AND I THINK IT’S SCALEABLE TO DO THE HUMAN GENOME. SO REALLY, THIS IS SOMETHING THAT WE’RE DEVELOPING THE IDEA TO DO THIS AND I THINK WE COULD HAVE TREMENDOUS INFORMATION YIELD AND SO THAT’S GOING TO BE A BIG FUTURE DIRECTION OF THE LAB. SO I THINK I’LL JUST STOP HERE AND TAKE QUESTIONS. [APPLAUSE]>>IF IF YOU HAVE QUESTIONS, PLEASE, THERE ARE MICROPHONES IN THE AISLES SO THAT PEOPLE CAN HEAR THE QUESTIONS WHO ARE WATCHING BY VIDEO, AND WE HAVE TIME FOR SEVERAL OF THOSE. SO PLEASE. WHILE PEOPLE ARE ARE GETTING TO THE MIKE N TERMS OF ASSAY THAT UH YOU SHOWED US WHICH LOOKS VERY NICE IN TERMS OF LOOKING FOR HOMO AND HETERO DIMERS BUT LOOKS LIKE IT COULD APPLY TO LIGAND RECEPTOR INTERACTIONS; HAVE YOU TRIED IT THAT WAY?>>ABSOLUTELY. IT’S ROBUST AND IT WORKS — THE AFFINITY ENHANCEMENT WE GET BY USING THE PENT MER, IT’S SO SENSITIVE THAT E WE DETECT INTERACTIONS OF MOLECULES THAT WE DON’T EVEN SEE EXPRESSION OF BY WESTERN BLOCK. SO IT’S THAT SENSITIVE.>>VERY GOOD TALK. CAN YOU GIVE US AN IDEA ON THE STATISTICS ON DATA SUCH AS B FACTORS AND RESOLUTION FOR ALL THE STRUCTURE YOU CALCULATED FOR WNT AND FRIZZLE?>>SORRY?>>SOME OF THE SPECIFIC DATA SUCH AS THE RESOLUTION THAT YOU OBTAINED B FACTORS — >>[LOW AUDIO] CIG STRUCTURES WERE ON THE SAME ORDER. THE OTHER STATISTICS LIKE B FACTORS AND THINGS ARE PERFECTLY NORMAL AND YOU CAN FIND THEM IN THE PAPERS.>>ALSO, WHAT HAPPENS IF YOU REMOVE THE FATTY ACID TAIL FROM WNT OR HOW SHORT CAN YOU GO WITH THAT FATTY ACID TAIL UNTIL YOU LOSE INTERACTION COMPLETELY?>>THIS IS THE PROBLEM WITH WNT BECAUSE THE WNT DOESN’T SECRET WITHOUT THAT FA ETY ACID ON IT. THERE’S A ENZYME CALLED PORCUPINE, IT’S AN EVOLUTIONARILY VERY CONSERVED ENZYME THAT’S RESPONSIBLE FOR ADDING THAT LIPID TO WNT AND IF THAT LIPID ADDITION SITE IS NOT THERE, WNT DOES NOT GET OUT OF THE ER.>>SO WHY IG DOMAIN ONLY? SEEMS TO BE THAT DEORPHAN STUFF, SEEMS TO ME THAT WOULD BE INSTRIN SICK DESIGN CAN BE APPLIED TO FOR EXAMPLE SCREEN FOR TCR LIGAND AS WELL; SO WHY JUST IG DOMAIN? AND MANY PROTEIN RECEPTOR LIGAND ARE NOT BOUND WITHIN IG DOMAIN, YOU KNOW VERY WELL; SO, FOR EXAMPLE, YOU KNOW, IG DOMAIN RECEPTORS FOUR WITH INTERACTION TCR AND SEED WHICH IS [INDISCERNIBLE] IG — SO.>>YEAH. SO, FIRST OF ALL, IG DOMAINS ARE THE MOST ARE THE LARGEST FAMILY OF STRUCTURAL CLASS OF CELL SURFACE ARE RECEPTORS. SO WE FOCUSED ON THEM. THIS WAS A PROOF OF CONCEPT EXPERIMENT TO REALLY ASK IF WE COULD DEORPHANIZE RECEPTORS ON A LARGE SCALE, AND SO IG SUPER FAMILY RECEPTORS, THEY’RE NOT HETERO DIMERS, WE JUST WANTED SINGLE POLLY TIDE CHANGES SO WE CHOSE IT OUT OF SIMPLICITY. HERE’S THE OTHER THING. WE CHOSE IT BECAUSE THE TOTAL NUMBER IS MANAGEABLE BECAUSE WHEN YOU DO A SCREEN LIKE THIS, YOU CAN’T DO PART OF IT. YOU HAVE TO DO THE WHOLE THING OR YOU SHOULDN’T EVEN TRY BECAUSE YOU’RE GOING MISS, AND SO THERE WERE TWO HUNDRED OR SO IG SUPER FAMILIES AND WE CALCULATED GIVEN THE PEOPLE WORKING ON THE PROJECT, WE’D HAVE THE BANDWIDTH TO HANDLE A 20,000 MATRIX ARRAY. SO LIKE THE HUMAN GENOME HAS ABOUT SIX HUNDRED IG SUPER FAMILY CONTAINING RECEPTORS. SO THAT WOULD BE ABOUT A FOUR HUNDRED THOUSAND ARRAY, UM, BUT, UH-UH UH, YOU KNOW, THIS IS EASILY SCALEABLE TO DO THAT. YEAH.>>IN TERMS OF THE ACTUAL MECHANISM OF FRIZZLED SIGNALING, DO YOU THINK WNT FINDING ACTUALLY CAUSES A CONFIRMATIONAL CHANGE OR BRINGS IT TOGETHER WITH OTHER SURFACE MOLECULES TO LEAD TO SMALL TRANSDUCTION?>>THAT’S A GOOD QUESTION BECAUSE THERE’S A LONG LITANY OF LITERATURE ABOUT THE COMPLEXITY OF THE FRIZZLED SIGNALING MECHANISM IN RESPONSE TO WNT AND WITH THAT ONE EXPERIMENT WE SHOWED WHERE IF UH YOU JUST BRING LRP TOGETHER WITH FRIZZLED IN A WAY THAT IS COMPLETELY UNLIKE WNT, YOU’LL GET A SIGNAL. SO I THINK IT’S PROBABLY SIMPLER THAN IT HAS BEEN MADE OUT TO BE.>>HAVE YOU TRIED TETHERING ARTIFICIAL MOLECULE TO ANOTHER INTERACTION DOMAIN TO BRING IT TOGETHER WITH ONE OF THE OTHER CORECEPTORS?>>WE HAVEN’T TRIED THAT YET, NO, BUT WE’RE TRYING A LOT OF THINGS NOW ALONG THIS.>>THANKS.>>YEAH.>>WELL I THINK THERE MAY BE OTHER QUESTIONS PEOPLE WOULD LIKE TO POSE TO THE SPEAKER. WE CAN ADJOURN TO THE NIF LIBRARY. LET’S THANK DR. GARCIA AGAIN.

Leave a Reply

Your email address will not be published. Required fields are marked *