WELCOME,
EVERYBODY.
IT'S A PLEASURE TO BE ABLE TO BE
HERE TODAY INTRODUCING HENRY
COLECRAFT FOR THE NEUROSCIENCE
SEMINAR SERIES.
HENRY'S BEEN A GOOD FRIEND FOR
MANY YEARS AND WE WERE
REMINISCING LAST NIGHT ABOUT HOW
WE MET AND IT WAS A LITTLE WHILE
AGO AND I'M NOT GOING TO SAY HOW
LONG BECAUSE I DON'T WANT TO
DATE MYSELF, BUT, HENRY HAS HAD
AN INTERESTING WANDERING CAREER,
HE WAS BORN IN GANNAA BUT THEN
MOVED TO ENGLAND FOR HIS
UNDERGRADUATE STUDIES AT KING'S
COLLEGE IN LONDON BUT THEN,
ACTUALLY AND I THINK THIS IS A
FEATURE OF MANY OF THE BEST
STORYTELLERS IN SCIENCE, HENRY
ACTUALLY RETURNED TO GHANA AND
TAUGHT HIGH SCHOOL FOR A FEW
YEARS BEFORE HE WENT TO GRADUATE
SCHOOL AND I THINK YOU SEE THAT
IN THE QUALITY OF HIS TALK.
IF YOU CAN TEACH SOMETHING TO
HIGH SCHOOL KIDS, YOU CAN TEACH
ANYBODY.
AND THEN, HENRY CAME TO THE U.S.
AFTER THAT TO GO TO -- AND DID
HIS GRADUATE WORK AT THE
UNIVERSITY OF ROUGH ATOM CHESTER
WHERE HE FOUND COLD AND THEN
WENT TO HOPKINS TO WORK WITH
DAVID [INDISCERNIBLE] AS A POST
DOC AND THAT'S WHERE HE MET UP
WITH THE CALCIUM CHANNELS HE'S
BEEN WORKING ON EVER SINCE.
HE THEN BECAME FACULTY IN
BIOENGINEERING DIRECTLY IN
HOPKINS BEFORE BEING RECRUITED
IN 2007, IS THAT RIGHT?
TO COLUMBIA WHERE HE'S BEEN IN
THE PHARMACOLOGY DEPARTMENT EVER
SINCE WORKING ON JUST REALLY
DOING BEAUTIFUL INSIGHTFUL WORK
ON CALCIUM CHANNELS AND THE
REGULATION, THEIR MECHANISM AND
AS YOU'LL SEE TODAY, REALLY HAS
ROBUSTLY MADE THE TRANSITION TO
INVESTIGATING WHAT CAN WE DO
PHARMACOLOGICALLY TO INTERVENE
IN THE SYSTEM AND TRY TO DREET
DISEASES OF IT AND THAT'S WHAT
WE WILL HEAR ABOUT TODAY BUT I
WILL NOT TRY TO ENUMERATE
ACCOMPLISHMENTS OR ANYTHING LIKE
THAT, IT'S EASY TO SEE HOW MUCH
HENRY HAS DONE BUT I JUST WANT
TO END BY TELLING YOU GUYS ABOUT
A QUOTE THAT I FOUND IN AN
INTERVIEW HENRY DID A FEW YEARS
AGO THAT WAS -- HE WAS TALKING
ABOUT HOW YOU GET INSIGHTS INTO
THESE CHANNEL MECHANISMS, MODEL
CITIZEN LEAK LECULAR MECHANISMS
IN GENERAL AND YOU KNOW WHAT YOU
HAVE DO IS MAKE YOURSELF REALLY
SMALL AND PRETEND YOU'RE IN THE
CELL WITH IT AND I THINK HENRY
HAS BEEN EXTREMELY SUCCESSFUL AT
MAKING HIMSELF SMALL BUT IN A
BIG WAY.
SO HENRY?
[ APPLAUSE ]
>> WELL, THANKS A LOT JOE.
FIRST TIME ANYBODY'S TOLL ME I'M
SMALL BUT I WILL TAKE THAT
ANYTIME OF DAY.
THANKS FOR THE INVITATION, IT'S
REALLY WONDERFUL TO BE HERE, JOE
WON'T SAY IT BUT I'VE KNOWN HIM
FOR OVER 20 YEARS AND ALSO A FEW
OTHER COLLEAGUESY HERE AND IT'S
REALLY GREAT AND I'VE ALREADY
LEARNED SOME REALLY GREAT
SCIENCE THIS MORNING.
SO I'M TYPICALLY TALKING TO
QUITE A BROAD AUDIENCE SO I
START MY TALKS OFF WITH 2
METAPHORS, WHICH REALLY CAPTURE
THE SCOPE AND UNIQUE CHALLENGES
OF A PROBLEM I GOT INTERESTED IN
THIS THE LAST 6 OR 7 YEARS OR
SO.
SO THE FIRST METAPHOR STARTs
IN THE CHESS BOARD, SO IN CHESS
1 WINS BY CAPTURING THE
OPPONENTS OF THE KING, BUT IN
ACTUALITY, THE MOST POWERFUL
PIECE IS THE QUEEN AND THAT'S
BECAUSE UNLIKE ANY OTHER PIECE,
IT CAN MOVE UNCONSTRAINED ACROSS
THE BOARD.
SO ON THE CHESS BOARD OF
BIOLOGICAL MOLECULES I WOULD
LIKE TO POSIT AS 1 PROTEIN THAT
IS PROBABLY THE MOST POWERFUL OF
THEM ALL WITH RESPECT TO YOUR
PARTICULAR PROTEIN THAT YOU'RE
INTERESTED IN, THAT PROTEIN IS 1
OF THE SMALLEST THERE IS, IT'S
CALLED CRUCIATE LIGAMENT CIBOL
WITTEN.
AND THE REASON WHY I SAY
UBIQUITIN IS THE QUEEN OF
BIOLOGICAL MOLECULES IS THAT IT
CONTROLS THE LIFE AND DEATH OF
EVERY OTHER PROTEIN.
SO ONCE A PROTEIN IS MADE, A
STEADY STATE CONCENTRATION IS
[INDISCERNIBLE] BETWEEN
UBIQUITINNIZATION, AND THE MOLL
CUE IS KEY WHICH DIRECTION THAT
GOES.
SO THERE ARE LITERALLY HUNDREDS
OF THOUSANDS OF DISEASES WHERE
WE HAVE TOO MUCH OR TOOLET OF A
PROTEIN SO IN PRINCIPAL THE
UBIQUITIN SYSTEM HAS A POTENTIAL
SOLUTION TO ALL THOSE KINDS OF
DISEASES BECAUSE IT SHOULD BE
POSSIBLE TO REGULATE THE HALF
LIFE OF A PROTEIN AND THAT'S A
STEADY STATE CONCENTRATION
BECAUSE IT'S A REGULATED AMOUNT
THAT A PROTEIN HAS BUT THAT'S
NOT REALLY THE WORLD WE LIVE IN.
AND UBIQUITIN IS NOT BEING
UTILIZED, IS NOT BEING USED TO
TARGET TOO MANY DISEASES AT THE
MOMENT AND THE REASON WHY HAS TO
DID WITH MAY SECOND METAPHOR.
SO CONSIDER A MAN WHO'S DYING OF
THIRST IN THE MIDDLE OF THE
OCEAN, THE IRONY IS THAT THE
APPARENT SOLUTION TO HIS PROBLEM
IS ALL AROUND HIM, IT'S WATER
BUT THAL CASE, THE WATER IS A
POISON CHALICE, IT HAS A LOT OF
SALT IN IT, IF YOU DRANK IT
WOULD SPEED UP DEHYDRATION IS
DEATH.
IN THE SAME WAY, UBIQUITIN WE
THINK IS A POTENTIAL KEY TO MANY
DISEASES BUT THE PROBLEM IS IT'S
EVERYWHERE, EVERY SINGLE PROTEIN
IS UTILIZING UBIQUITIN TO
MAINTAIN HOMEOSTASIS.
THE CHALLENGE BECOMES HOW DO YOU
TARGET A PROTEIN AND SPECIFICITY
WITHOUT DISTURBING THE WHOLE
GLOBAL PROTEOME THAT WOULD THEN
BE POTENTIALLY TOXIC?
SO WHAT I WOULD LIKE TO DO, THIS
OPPORTUNITY TO TALK ABOUT 3
STORIES THAT REALLY POINT TO OUR
WAYS TO DEAL WITH THAT ESSENTIAL
CHALLENGE AND TRY TO MAKE THE
UBIQUITIN SYSTEM IN A WAY MORE
TRACTABLE FOR DESIGNER
THERAPIES.
AND THE PARTICULAR PLAY GROWBD I
PLAY IN IS ION CHANNELS SO THE
STORY OF THE ION CHANNEL IS
CENTRIC, BUT THE UBIQUITIN
SUGGESTS REGULATES EVERY PROTEIN
AND THIS COULD BE APPLIED TO ANY
PROTEIN AS L. SO THE FIRST STORY
STARTS WITH THE REALIZATION WE
HAVE, THERE ARE SEVERAL DISEASES
THAT CAN BE CHARACTERIZED AS
RARE, THEY'RE DEVASTATING THINGS
LIKE ARRHYTHMIAS THAT HAVE
SUDDEN CARDIAC DEATH, CYSTIC
PHOBEROSIS THAT OCCURS IN THE
LUNGS AND EPILEPSY AND THE LIST
GOES ON.
THESE ARE VERY DIFFERENT
DISEASES AFFECTING DIFFERENT
ORGAN SYSTEMS AND AT FIRST
GLANCE IT DOESN'T SEEM LIKE
THERE'S MUCH THAT CONNECTS THEM
BUT THAT COULD BE POTENTIALLY
COMMON SOLUTIONS FOR FURTHER,
BUT WHAT WE REALIZE UPON DEEPER
REFLECTION IS THAT MANY
INSTANCES OF THESE DISEASES, ARE
DUE TO MUTATIONS IN ION
CHANNELS, THAT CAUSE A DECREASE
IN THE FUNCTIONAL EXPRESSION,
OKAY?
SO FROM THAT PERSPECTIVE, THESE
DISEASES HAVE THE SAME VERY
DIFFERENT DISEASES HAVE THE SAME
COMMON BIOLOGICAL PROBLEM AND
THESE SIMPLE BIG IDEA THAT
EMANATES FROM THAT IF WE COULD
FIGURE OUT A WAY TO STABILIZE
THESE PROTEINS YOU COULD HAVE
SOMETHING THAT COULD BE
GENERALIZABLE TO MANY DEFINITE
DISEASES.
SO THE ION CHANNELS THAT WE'RE
INTERESTED IN, WORK AT THE CELL
SURFACE, AND THE SURFACE DENSITY
IS REALLY REGULATED BY 3
PROCESSES ISSUES RATE OF
DELIVERY, RATE OF REMOVAL AND
RATE OF DEGRADATION OF THE OTHER
PROTEIN AND FOR WILD-TYPE
PROTEINS THE UBIQUITIN TAGGING
ON TO THE PROTEIN DICTATES WHERE
THE EQUILIBRIUM RELIES, SO IF
YOU HAVE THIS, YOU GET THIS TO
FIRST.
SO THE REALIZE WE HAD WAS THAT
THERE WERE MANY MUTANT ION
CHANNELS THAT CAUSE THESE LIFE
THREATENING DISEASES, WHERE THE
MUTATION CAUSED THE CHANNEL TO
BE TRAPPED INSIDE THE CELL, SO
IN THAT WAY, IT'S VERY SIMILAR
TO A WILD-TYPE CHANNEL WITH A
LOT OF UBIQUITIN ON IT, SO THE
SIMPLE HYPOTHESIS WE HAD THEN
WAS THAT WE COULD POTENTIALLY
STRIP UBIQUITIN FROM MUTANT
CHANNELS AND CAUSE THEM TO BE
STABILIZED AND GET TO THE CELL
SURFACE, AND POETIC TEBTIA WILY
RESCUE FUNCTION.
-- POTENTIALLY RESCUE FUNCTION.
I'VE ALREADY TOLD YOU THE
ESSENTIAL CHALLENGE TO DO THAT,
YOU HAVE TO DO THAT AT
SPECIFICITY SO YOU DON'T HAVE
TOXICITY ASSOCIATED WITH THAT.
SO THE WAY WE SOLVE THAT PROBLEM
IS TO COME UP WITH A NEW TYPE OF
MOLECULE, WE CALL THESE
ENGINEERED DECIBOL WITTEN ACES
AND THEY'RE BI VALENTINEDENT
MOLECULES THAT HAVE A TARGET
MOEITY, A NANO BODY THAT ALLOWS
TO YOU HAVE SPECIFIC CELLS OF
CHOICE.
WE FELL THAT CATALYTIC DOMAINS
UBIQUITIN AS OR DUBS, SO WITH
THAL SIGN FEATURE YOU HAVE
SPECIFICITY BY THE NANO BODY,
THE ISS IS MODULAR, YOU SHOULD
BE ABLE TO SWAP OUT NANO BODIES
FOR DIFFERENT TARGETS AND WE
EXPECTED TO BE HIGHLY POTENT
BECAUSE OF THE CATALYTIC
ACTIVITY OF THE ENZYME.
SO THIS IS THE CARTOON CONCEPT,
COULD WE GET THIS TO WORK IN
CELLS?
SO SCOTT CANON IS THE STUDENT
WHO DEVELOPED THE IDEA IN THE
LAB, TO SEE THE PROOF OF CONCEPT
OF THE TECHNOLOGY, HE CAME UP
WITH A BIOCHEMICAL ASSAY TO TEST
THAT.
SO IN HEX CELLS WE CAN EXPRESS A
QKKCM ATTACHED TO YFP AND THIS
IS A POTASSIUM CHANNEL AND SO IF
YOU CO-EXPRESS IT THE CHANNEL
GETS UBIQUITINNATED AND GETS
ENDOSIGNIFYITOSED AND A LOT OF
IT GETS DEGRADED EMPLOY SO NOW
WHAT WE'RE ABLE DO THEN IS WE
COULD THEN CO-EXPRESS AN NDUB, A
NANO BODY THAT TARGETS THE YFP,
BRINGS THE DUB TO THE TARGET
DECIBOL WITTENATEED AND WE COULD
GET A REVERSAL OF THAT PROCESS.
SO HERE'S BIOCHEMICAL PROOF OF
THE CONCEPT.
WE EXPRESS THE CHANNEL Q1, AND
THE CONTROL CONDITIONS WITH THE
NAKED NANO BODY.
YOU PULL DOWN THE CHANNEL AND
PROBE IT, YOU FIND 4 BANDS
REPRESENTING THE MONOMER,
TRIEMER, TETRAMERIC SPECIES OF
THE CHANNEL.
IF YOU CO EXPRESS NET FOIL THE
CHANNEL, THE DENSITY OF THOSE
GOES DOWN AS THE CHANNEL IS
ENDOSIGNIFYITOSED AND DEGRADED.
SO WHAT WE CAN THEN DO IS STRIP
THIS BLOT AND PROBE FOR
UBIQUITIN AS YOU WOULD EXPECT,
UNDER BASE LINE CONDITIONS
THERE'S A BIT MORE, AND THAT
GETS MORE IN THE E-3 LIGASE, SO
THIS ARM OF THE PATHWAY IS
ESTABLISHED AND NOW WE CAN HAVE
AN NDUB AND SEE WHAT HAPPENS.
WHEN WE DO THAT YOU RESTORE THE
PROTEIN EXPRESSIONS AND THAT'S
ACCOMPANIED BY THE UBIQUITIN
PROTEIN.
SO BIOCHEMICALLY THIS APPEARS TO
FUNCTION.
WHAT WE NEEDED TO KNOW THEN WAS
WHETHER THESE DEUBIQUITINNATED
CHANNELS GET TO THE CELL SURFACE
ARE FUNCTIONAL?
TO DO THAT WE DEVELOPED A FLOW
CYTOMETRY ASSAY, THAT ALLOWS TO
US TO SIMULTANEOUSLY LOOK THEA
SURFACE CHANNELS USING A
FLUORESCENT TOXIN THAT BINDS IN
THE EPITOPE THAT'S ENGINEERED
INTO THE SURFACE OF THE ION
CHANNEL, AND THEN WE HAVE THE
YFP TO GIVE US A MEASURE OF THE
TOTAL PROTEIN EXPRESSION, OKAY.
SO WHEN YOU APPLY THAT TO FLOW
CYTOMETRY, WHAT YOU CAN SEE IN
THE CONTROL CONDITIONS IS THAT
YFP POSITIVE CELLS HAVE A LOT OF
REGINALSOT CELL SURFACE, THE
CHANNELS ARE RIGHT AT THE
CHANNEL MEMBRANE, THE DENSITY
GOES DOWN AS WELL AS YFP
INTENSITY GOES DOWN AS PROTEIN
IS DEGRADED AND
ENDOSIGNIFYITOSED.
SO NOW WHEN WE ADD THE NDUB, WE
FIEBD A RESCUE OF BOTH THE
SURFACE DENSITY AND THE TOTAL
PROTEIN EXPRESSION.
SO PROEN TOOS DO MAKE IT TO THE
CELL SURFACE, TO DETERMINE
WHETHER THEY'RE FUNCTIONAL WE
USE THE PATCH CLAMP, WHOLESALE
PATCH CLAMP TECHNIQUE.
CONTROL CHANNELS EXPRESS ROBUST
OUTWARD OCCURRENCE, CO EXPRESSED
BEFORE ALL THE CHANNELS ARE GONE
AND WHEN YOU ADD THE ENDUB, YOU
RESCUE THOSE CURRENTS, SO THE
DECIBOL WITTENATEED CHANNELS ARE
FINE.
DID A NUMBER OF CONTROL
EXPERIMENTS TO FIND OUT THAT
THIS REQUIRED IT TO BE TARGETED
TO THE END OF CHOICE AND THOSE
ARE TARGETABLE EFFECT CHES.
SO WITH THAT ESTABLISHED WE ARE
READY TO APPLY THE SYSTEM TO A
RARE DEC.
THE FIRST 1 WE LOOKED AT IS
[INDISCERNIBLE] SYNDROME, 1 IN 2
BIRTHS ASHING THE 400,000 SUDDEN
DEATH KNOW DEATH CASES IN THE
U.S. AND MUCH MORE BEYOND THE
U.S.
SO LOSS OF FUNCTION MUTATIONS IN
THE CHANNEL HAVE BEEN TALKING
ABOUT CALCIUM K1, COME FROM
THERE 35% OF THESE LQTS CASES,
SO THIS CHANNEL GIVES RISE TO
SLOWLY ACTIVATED POTASSIUM
CURRENT THAT'S REALLY IMPORTANT
FOR THE REPOLARIZATION PHASE OF
THE CARDIAC ACTION POTENTIAL SO
WHEN YOU HAVE LESS OF IT, YOU
HAVE A PROLONGATION OF THE
ACTION PROTENTIAL THAT SHOWS UP
AS A PROLONGATION OF THE LQTS
CARDIOGRAM AND THE BAD THING
ABOUT IT IS THAT PEOPLE WHO HAVE
ARE PREDISPOSED TO GETTING
LETHERAL VENTRICULAR ARRHYTHMIA
WHEN THEY TWERICIZE, SO IT TURNS
OUT MOST OF THOSE MUTATIONS AND
YOU CAN USE ENDUBS TO STRIP THEM
FROM THESE CHANNELS AND CAUSE
THEM TO BE RESCUED.
SO WITH THAT IN MIND, WE MADE A
NUMBER OF PATIENT MUTATIONS AND
USING THE ASSAY TO LOOK FOR
SURFACE TRAFFICKING, WE ASSESS
THE IMPACT OF THOSE MUTATIONSOT
SURFACE SENSITY OF THE CHANNELS,
SO THE DOTTED LINE REPRESENTS
WHAT YOU GET WITH WILD-TYPE MANY
OF THOSE CHANNELS CO EXPRESS
WITH THE NCI--AND NANO BODY,
HAVE A DEFICIENCY IN TRAFFIC TO
THE CELL SURFACE.
HOWEVER, WHEN WE ADD THE ENDUB,
WE ARE ABLE TO RESCUE MANY OF
THEM, NOT ALL OF THEM BUT A
LARGE FRACTION OF THEM TO
ESSENTIALLY WILD-TYPE LEVELS OF
THE SURFACE DENSITY.
IF WE -- AND YOU CAN LOOK TO SEE
THAT PICTORIALLY HERE, SO HERE
WE ARE STAINING THE SURFACE
CHANNELS WITH THE FLUORESCENT
TOXIN, IN THIS MUTANT, THAT
STANDARD IS GONE, BUT YOU CO
EXPRESS WITH THE ENDUB AND YOU
GET IT RIGHT BACK.
ALL RIGHT, SO WE'VE BEEN ABLE TO
RESCUE, A CHANNEL THAT'S TRAPPED
INSIDE THE CELL, THE NEXT
CHALLENGE IS WHETHER THOSE
CHANNELS WERE FUNCTIONAL AS THEY
GET TO THE CELL SURFACE.
SO USING ELECTROPHYSIOLOGY HERE
IS WHAT YOU GET WITH THE
WILD-TYPE CURRENT AGAIN.
HERE'S 1 OF THOSE MUTANTS ARE
591 H, YOU CAN SEE THAT THE
CURRENT IS ALMOST TOTALLY
ABOLISHED, ML277 IS A SMALL
MOLECULE AGONIST THAT IF YOU
HEAD ADD THE IN YOUITATION, YOU
INDICATE THAT SOME OF THE
CHANNELS GET TO THE SURFACE, SO
HERE'S WHAT HAPPENS WHEN WE CO
EXPRESS THE MUTANT, AND YOU GET
A 50% RESCUE OF THE CURRENT.
THIS IS ACTUALLY QUITE
INTERESTING BECAUSE IF YOU WE
LEAK AT THE SURFACE DENSITY, IT
DOES RESCUE A HUNDRED PERCENT IS
SO WHAT THIS VEALS IN THE
TRAPPED DEFICIENCY, THE MUTATION
CAUSES A [INDISCERNIBLE], AND
DEFICIT AS L. HOWEVER, NOW ON
TOP OF THAT IF WE ADD THE SMALL
MOLECULE AGONIST, YOU CAN GET A
SUPER RECUSE OF THE
[INDISCERNIBLE], AND YOU CAN
ASHES TACH THE SMALL MOLECULE TO
GET WILD-TYPE CURRENTS BACK.
SO THIS WAS ENCOURAGING.
IN ORDER TO SEE WHETHER THIS
COULD HAVE A POTENTIAL
THERAPEUTIC POTENTIAL, WAS IT
IMPORTANT TO GO FROM THESE MODEL
SYSTEM OF HEX CELLS INTO THE
HEART ITSELF.
WE COULDN'T TEST THE EFFICACY OF
THESE IN MICE, IN MICE BECAUSE
THEY ARE ACTUALLY POTENTIALS ARE
TOO SHORT, SO THIS POTASSIUM
CURRENT ACTUALLY DOESN'T PLAY A
ROLE IN THE REPOSEARIZATION OF
THAT, SO THE MODEL WE CAME UP
WITH WITH WAS TO TAKE A GINNY
PIG MONOCYTE WHICH DOES EXPRESS
ENDOGENOUS K1 AND WE CAN USE AN
ADENOVIRUS TO EXPRESS MORE ON
THAT, IT COMBINES WITH THE
ENDOGENOUS 1.
IF YOU DO A ROUND PROTOCOL ON
THESE GINNY PIG MICE, YOU GET
THIS WAVE FORM OF CURRENT AND
THE ICS, CURRENT, CONTRIBUTE TO
THE POTASSIUM CHANNEL IS THIS
LATE PHASE OF THE WAVE FROM --
AND THIS THE NORMAL LOOKING
POTENTIAL YOU CAN RECORD IN
THESE MICE.
SO THEN WHAT WE CAN DO THEN IS
TO USE AN ADENOVIRUS TO EXPRESS
THE MUTATION, AND THIS MIMICS
THE AUTOSOMAL DOMINANT NATURE OF
THIS DEC IN HUMANS SO THE MUTANT
WILL MIX WITH ENDOGENOUS 1 AND
IF YOU LOOK AT CURRENT, CAN YOU
SEE IT'S ABOUT HALF, SO IT HAS A
DOMINANT NEGATIVE EFFECT ON THE
CURRENT.
SO WHAT -- AND THE RESULT OF
THAT IS THIS CLASSICAL SIGNATURE
OF LONG QT SYNDROME,
PROLONGATION OF THE ACTION
POTENTIAL IN THIS SYSTEM, AND SO
WE CAN ADD AN ENDUB TO THIS AND
SEE IF WE CAN RESCUE THE CURRENT
POETIC TEBTIAL AND THE ANSWER IS
YES.
SO THIS RESCUES THE OUTWARD
CURRENT AND NORMALIZES THE
POTENTIAL DURATION, SO PREDICT
THAT IF YOU DID THIS IN THE
HUMAN THAT THAT WOULD BE
BENEFICIAL FOR THE LONG QT
SYNDROME.
SO 1 OF THE THINGS THAT WE WERE
MOST EXCITED ABOUT, THE
TECHNOLOGY IS THE IDEA THAT IT
SHOULD BE GENERALLY APPLICABLE
TO BASICALLY ANY PROTEIN.
AND SO TO SEE WHETHER THAT'S THE
CASE FOR THE ION CHANNELS, WE
LOOKED AT ANOTHER ION CHANNEL
DISEASE, THIS CASE,
CYSTIC FIBROSIS, FOR JUST REALLY
THE POSTER CHILD OF ION CHANNEL
MUTATION CAUSING A DEC.
SO IN THIS CASE, THE MUTATION
OCCURS IN THE CHLORIDE CHANNEL,
THAT LINES THE AIR WAY
EPITHELIAL CELLS, IN THE AIR
WAYS, THIS CHANNEL NORMALLY
ALLOWS CHLORIDE TO COME OUT OF
THE EPITHELIAL CELLS AND THAT
DRAGS WATER AND THAT'S VERY
IMPORTANT FOR HYDRATING THE
AIRWAYS, WHEN YOU HAVE A LOSS OF
FUNCTION MUTATION, YOU GET LESS
WATER COMING UP SO YOU GET A
THICK BUILD UP OF MUCUS IN THE
AIRWAYS AND THAT TRAPS BACTERIA,
CAUSES ROUNDS OF INFECTION AND
SO FORTH THAT ULTIMATELY DESTROY
THE LUNGS.
SO PEOPLE SUFFERING FROM THIS
HAVE A LOW QUALITY OF LIFE AND
ALSO A SHORTER LIFE SPAN.
SO WE ASK WHETHER ENDUBS COULD
BE USED TO HELP OUT
CYSTIC FIBROSIS.
IN ORDER TO DO THAT WE GENERATED
NANO BODIES TO THE CFTR CHANNEL,
WE PURIFIED THE NBD1 DOMAIN AND
USED IT AS BAIT TO FISH OUT
BINDERS FROM A SYNTHETIC YEEOF
THE NANO BODY LIBRARY THAT
CONTAINS A HUNDRED MILLION
UNIQUE NANO BODIES AND AFTER
ROUNDS OF THEM, 2 OR 3 ROUNDS OF
THE SELECTION, WHAT YOU CAN SEE
HERE IN THIS FLOW CYTOMETRY, FAX
EXPERIMENT IS YOU GET AN
EVOLUTION OF BINDERS RIGHT HERE,
SO WE HAVE A NUMBER TO THE
CFTRAD LEAST TO THE BD1.
TO DETERMINE IF IT BINDS, WE USE
A FRET ASSAY, AND THIS SHOWS
THAT THE BINDER THAT BINDS THE
FULL PROTEIN COMPARED TO A
NEGATIVE CONTROL.
OKAY.
SO WITH THE BANDER WE'RE ABLE TO
ARM THAT AND CONVERT IT INTO AN
ENDUB AND SEE IF WE COULD RESCUE
METRICSITANCY OF CRT CFTR
CHANNELS.
THE ATIN03 K IS THE ABOUT THE
THIRD OR FOURTH MOST COMMON
MUTATION.
WHAT ARE YOU LOOKING AT HERE, IN
BLACK ARE THE WILD-TYPE CURRENTS
WITH THE WILD-TYPE RELATIONSHIP,
WITH WILD-TYPE AND CHLORIDE
CHANNEL CFTR, IN RED ARE THE
MUTANT.
SO YOU CAN SEE THAT AT ANY
[INDISCERNIBLE].
THEY HAVE MINIMUM CURRENT GOING
THROUGH THEM.
ANOTHER IMPORTANT DIFFERENCE IN
THE MUTATIONS IS THAT THERE'S
SMALL MOLECULE MUTATION THERAPY
FROM VERTEX PHARMACEUTICALS THAT
HAVE BEEN APPROVED FOR THE F508
DEL, BUT IS INEFFECTIVE FOR THE
N1303 K.
SO WHEN YOU ADD TRI TRIKAFTA,
YOU GET A SMALL INCREASE IN THE
CURRENT, NOWHERE NEAR THE
WILD-TYPE BUT SUDDENLY
RATIONALIZES WHY THE PATIENTS
FEEL BETTER ON THIS DRUG.
THE N1303 K DOES MARGINALLY
WORSE THAN THIS AND THAT'S ALSO
CONSISTENT WITH WHY THE PATIENTS
DON'T DO AS WELL.
WHAT WE'RE REALLY EXCITED ABOUT
IS WHEN WE COMBINED THE
TRIKAFTA, WITH THE END OF IT,
YOU GET A WILD-TYPE, THE 1
THAT'S BEEN APPROVED FOR FDA FOR
TRIKAFTA, AS WELL AS WHAT HASN'T
AND THAT'S SUFFICIENT TO CAUSE A
RESCUE OF THOSE CURRENTS.
AND SO WE'VE BEEN USING THESE
ENDUBS IN VARIOUS DIFFERENT ION
CHANNELS AS WELL AS OTHER
DEFINITE PROTEINS, AND YOU KNOW,
IT'S VERY HARD TO -- SO FAR WE
HAVEN'T FOUND ANYTHING THAT IT
DOESN'T WORK FOR.
SO I'LL LEAVE IT THERE.
OKAY.
SO I'LL NOW GO ON TO A SECOND
STORY, THAT WE'VE BEEN WORKING
ON AND THAT TO SET UP THAT
STORY, I WILL IMAGINE, ASK YOU
TO IMAGINE BEING PUT INTO THE
MIDDLE OF A VERY COMPLICATED
CITY AND BEING TOLD TO GET FROM
POINT A TO POINT B WITHOUT THE
AID OF A MAP OR A GPS.
FOR ME, EVEN WITH A GPS, IT
MIGHT BE A PROBLEM, OKAY IN BUT
THIS SAME SORT OF PROBLEM IS
WHAT I THINK IS FACED BY ION
CHANNELS THAT I MADE INSIDE THE
CELL AND REALLY HAVE TO NAVIGATE
A VERY COMPLEX INTRACELLULAR
ENVIRONMENT IN ORDER TO GET TO
THE CELL SURFACE WHERE THEY
FUNCTION.
SO WHAT WE'VE BEEN INTERESTED IN
OR 1 OF THE QUESTIONS WE'VE BEEN
INTERESTED IN ADDRESSING IS YOU
KNOW WHAT ARE THE DIRECTIONS OR
SIGNALS THAT ENABLE ION CHANNELS
TO REALLY TRAVERSE THIS VERY
COMPLICATED SYSTEM TO GET TO THE
CELL SURFACE AND WE WILL USE
THIS TOOL THAT I'VE ALREADY
SPOKEN TO YOU ABOUT THE CHANNEL
WHERE WE ARE AN EPITOPE TAG, AND
LOOK AT ALSO THE YFP SO WE CAN
LOOK AT THE TOTAL PROTEIN
EXPRESSION.
SO IF YOU EXPRESS THIS ENGINEER
CHANNEL INSIDE THE HEX CELL AND
LOOK FOR WHERE IT'S DISTRIBUTED,
YOU FIND IT IN IN OTHER PLACE
WHERE YOU FIND IT, IN THE ER,
GOLGI, ENDOSTUDIES OF MULTIPLE
ENDOCRINES AND LIES SOSOMES SO
IT GOES THROUGH THE TRAFFICKING
PATHWAY.
AND THE QUESTION WE'RE
INTERESTED IN IS WHAT ACTUALLY
DICTATES THE ABILITY OF THOSE
CHANNELS TO REALLY GO THROUGH
THOSE PATHWAYS, RIGHT?
AND THE HYPOTHESIS WE HAD WAS
THAT THE UBIQUITIN SUGGESTS WAS
INVOLVED IN THAT, BECAUSE
UBIQUITIN IS MOST WELL KNOWN FOR
ITS ROLE IN PROTEIN DEGRADATION,
BUT THERE ARE INDICATIONS IT
COULD POTENTIALLY ALSO BE
INVOLVED IN TRAFFICKING AND SO
FORTH.
SO HOW CAN THAT HAPPEN, THE SAME
SIGNAL BE UTILIZED FOR THESE
MANY DIFFERENT EFFECTS?
WELL 1 THING THAT WE -- THAT
COULD BE THE CASE IS THAT
UBIQUITIN IS NORMALLY ATTACHED
TO LYSINE SUBSTRATES BUT IT
TURNS OUT THE UBIQUITIN ITSELF
HAS 7 DISTINCT LYSINEs AND
THOSE COULD BE UBIQUITINNATED AS
WELL.
SO DEPENDING ON THE TYPE OF
UBIQUITIN THAT'S ATTACHED TO A
PARTICULAR LYSINE, CAN YOU GET
UBIQUITIN THAT LOOK VERY
DIFFERENT IN TERMS OF SIZE AND
SHAPE AND SO COULD BE RECOGNIZED
DIFFERENTLY WITHIN THE CELL.
SO K48 IS THE CANNONICLE
DEGRADATION SIGNAL WHERE AS
CASES FOR EXAMPLE, STARTED TO BE
NONDEGRADATION
GRATES--GRATUEIDATIVE AND
FUNCTIONS.
AND THE SYSTEM GETS MORE
COMPLICATED BECAUSE MASS SPEC
HAS INDICATED YOU CAN FORM
DIFFERENT POLYUBIQUITIN CHAINS
THAT HAVE VERY DIFFERENT
ARCHITECTURE SYSTEMS AND SO
FORTH SO YOU CAN IMAGINE THAT
MOST OF THE CELLS THAT ARE --
MOST OF THE PROTEINS INSIDE OF
CELL REALLY HAVE A TON OF
UBIQUITIN ON THEM AND WE HAVE
ABSOLUTELY NO IDEA THE
FUNCTIONAL CONSEQUENCES OF THIS.
SO OUR HIGHWAY PUTTING SIS WAS
THAT THIS SOMEHOW WAS INVOLVED
IN THE REGULATING ION CHANNELS
AND THAT'S THE THING THAT WE
WILL INVESTIGATE.
SO TO ADDRESS THAT, THE FIRST
THING WE ASKED IS WHETHER THE
CHANNEL EXPRESSED IN HEX CELLS
DOES HAVE ANY POLYUBIQUITIN ON
IT, SO WE -- WE PULL DOWN THE
CHANNEL EXPRESSING HEX CELLS,
AND THEN WE COULD CUT OUT THE
MONOMERAND THE DIME MERRIC
SPECIES AND HAVE THEM IN MASS
SPECT AND IN ADDITION TO THE
PEPTIDES REPRESENTING THE
CHANNEL, WHAT WE ALSO SAW IN
THERE WERE PEPTIDES THAT
CORRESPONDED TO UBIQUITIN AND.
UBIQUITIN DEPENDING ON THE
LYSINE THAT IS UBIQUITINNATED IT
LEAVES BETWEEN A DISCIPLINARY
GLAISIN SIGNATURE, SO BASED ON
WHERE THE DISCIPLINARY GLIESIN
IS ON THE LYSINES, YOU COULD
FIGURE OUT WHICH 1S WERE
UBIQUITINNATED.
SO WHAT WE DID NOW IS WAS ON THE
UBIQUITIN CALCIUM CHANNEL WE
FOUND OUT 40% OF REPRESENTED
K48, ABOUT 24%, WAS THE K AND
THERE WAS A MINOR'S SPECIES OF
ABOUT 4%.
SO K48 IS THE CANNONICLE
DEGRADATION SIGNAT SIGNATURES E
LITERATURE.
ALL RIGHT, SO, THE QUESTION NOW
BECOMES, SO, WE DO HAVE
POLYUBIQUITINNATION OF THE
CHANNEL.
WHAT DOES IT DO?
THERE'S NO WAY THAT RIGHT NOW
THAT YOU CAN ADDRESS THAT IN A
LIVE CELL.
BUT WE THOUGHT WE COULD
POTENTIALLY EXTEND THE ENDUB
APPROACH THAT WE THEN DEVELOPED
TO BEGIN TO ADDRESS THOSE KINDS
OF QUESTIONS.
AND WE'RE GOING TO MAKE USE OF 7
TYPES OF DECIBOL WITTEN ACES
THAT HAVE EXQUISITE LINKAGE
SPECIFICITY AND THE ABILITY TO
HIDE ROLLIZE THE POLYUBIQUITIN
CHAINS.
SO FROM [INDISCERNIBLE]'S LAB,
YOU HAVE DEUBIQUITIN AISS AND SO
THE IDEA WAS THAT WE COULD NOW
HIJACK SOME OF THOSE LINKAGE
SPECIFIC DEUBIQUITIN ASES AND
THAT'S HOW YOU GET THE SELECTIVE
ENDUBS.
SO WE GENERATE THESE MOLECULES,
FIRST WE ASKED WHETHER THEY HAD
ANY EFFECT ON THE BIOCHEMICAL
UBIQUITIN SIGNATURE OF THE PULL
DOWN CHANNEL.
SO COMPARED TO THE BASE LINE
CONTROL, WE SAW THAT ALL THESE
PUTATIVE LINKAGE SELECTIVE
ENDUBS DECREASE THE UBIQUITIN
SIGNAL ON THAT ON THE CHANNEL
AND SO THIS INDICATE THAD THEY
HAD SOME ACTIVITY, THEN WE WERE
SURPRISED THAT THEY ALL SEEM TO
HAVE PRETTY PROBUST ACTIVITY
GIVEN THAT K48 WAS THE MOST
DOMINANT 1.
THAT'S MEDIATED BY THIS 1.
WHEREAS [INDISCERNIBLE] THE
MINOR SPECIES BUT HOWEVER THEY
ALL SEEM TO BE VERY EFFECTIVE IN
HIDE ROLLIZING THE UBIQUITIN
FROM THE PULL DOWN CHANNEL.
ONE POTENTIAL WAY THAT COULD
ADDRESS THAT -- THAT COULD
EXPLAIN THAT THAT THERE'S A LOT
OF BRANCHING IN THE UBIQUITIN
CHAIN ANDS THAT'S SOMETHING WE
CAN GET TO.
HOWEVER, SO, BASIC LE USING THE
BIOCHEMICAL SIGNATURE, WE
COULDN'T REALLY TELL WHETHER
THERE WAS A DIFFERENCE AND HOW
THESE THINGS WERE HYDROLLIZING
FROM THE CHANNEL.
BUT NOW WE CAN LOOK AT
FUNCTIONAL OUTPUTS TO SEE THE
DIFFERENT LINKAGES HAVE
DIFFERENT EFFECTS ON THE
EXPRESSION AND THE TRAFFIC OF
THE CHANNEL, SO FESTER WE LOOK
AT YFP SIGNAL AND THAT GIVES YOU
AN INDICATION OF THE TOTAL
AMOUNT OF PROTEIN THAT'S
EXPRESSED.
SO IF WE START FROM THE RIGHT
HAND SIDE, THIS IS ENDUB THAT
HIDE ROLLIZES ALL UBIQUITIN
CHAINS AND WHEN YOU DO THAT YOU
GET AN INCREASE IN THE AMOUNT OF
PROTEIN EXPRESS TD.
NOT SURPRISING WHEN YOU FIND OUT
THAT UBIQUITIN STABILIZES THE
ABILITY OF THE PROTEIN.
BUT WHAT SURPRISING WAS WHICH
LINKAGES WERE IMPORTANT.
SO THE DOMINANT 1 K48 WHICH IS
CANONICALLY RELATED TO PROTEIN
DEGRADATION, THAT HAD ABSOLUTELY
NO EFFECT ON THE SEPARATE OF THE
PROTEIN, NOR DID THE SECOND MOST
DOMINANT 1 K63 REMOVAL.
THE 1S THAT HAVE THE MOST IMPACT
ON THESE, GREATER PROTEIN WITH
THE MINOR SPECIES, THE K11 AND
K29, K33.
SO THAT WAS A FIRST SURPRISE
LOOKING AT THE TOLL AT PROTEIN
CONCENTRATION.
NEXT WE COULD LOOK AT WAS THE
SURFACE DENSITY OF THE CHANNELS
ISSUES THE ABILITY OF THE
CHANNELS TO GET TO THE CELL
SURFACE AND SO STARTING ONCE
AGAIN FROM THE RIGHT HAND SIDE,
EVEN THOUGH YOU GET MORE
PROTEINS BY REMOVING UBIQUITIN,
THAT DOESN'T NECESSARILY
TRANSLATE INTO MORE OF IT
GETTING TO THE CELL SURFACE.
SO UBIQUITIN IS NOT ONLY
IMPORTANT FOR THE STABILITY, BUT
POTENTIALLY COULD BE IMPORTANT
FOR THE TRAFFICKING OF THE
CHANNEL.
SECOND SURPRISING WAS TO -- WHEN
WE REMOVED THE K48, WE GOT LESS
CHANNELS ON THE CELL SURFACE.
K63 WAS PARTICULARLY NEUTRAL BUT
ONCE AGAIN WE'RE MOVING K11, AND
K29, PROMOTED THE SURFACE
TRAFFICKING OF THE CHANNEL.
SO ALL THESE INDICATED THAT
DIFFERENT LINKAGES WERE
POTENTIALLY INVOLVED IN ASPECTS
OF PROTEINS AND STABILITY AS
WELL AS TRAFFICKING ACCIDENT SO
THESE ARE STEADY STATE
MEASUREMENTS OF THE SURFACE
DENSITY SO WE COULD ALSO ADOPT
OUR FIRST TO LOOK AT THE RATE OF
FORWARD TRAFFIC AS WELL AS RATE
OF REMOVAL AND WHEY DID THAT WE
SAW A VERY DIFFERENT INTEREST IN
THEM AS WELL, SO LOOK AT THE
RATE OF FORWARD TRAFFICKING, THE
BLACK REPRESENTS THE CONTROL
CASE AND WHAT WE SAW WAS THAT
MOVEMENT IN THE MINOR SPECIES,
PROMOTED THE RATE OF FORWARD
TRAFFICKING, SO YOU GET MORE OF
THE CHANNELS GETTING TO THE CELL
SURFACE, REMOVING ALL UBIQUITIN
ACTUALLY HAD A -- YOU GOT A
PROFOUND LOSS OF THE ABILITY OF
THE CHANNEL TO TRAFFIC TO THE
CELL SURFACE.
THE OTHER THING WE COULD LOOK AT
WHAT WAS THE RATE OF REMOVAL
CHANNELS THAT ARE ALREADY AT THE
CELL SURFACE, AND ONCE AGAIN,
THEY WERE REMOVING DIFFERENT
LINKAGES AND HAD DIFFERENT
EFFECTS ON THEM, ON THE ABILITY
OF TURN OUT TO BE
UNDERSIGNIFYITOSE.
PUTTING ALL THAT TOGETHER WITH
THE DATA I DON'T HAVE TIME TO GO
THROUGH, CAME UP WITH THIS
EXCITING MODEL FOR HOW UBIQUITIN
ARE AFFECTING THE TRAFFICKING
PATHWAY.
SO MOST OF THE CHANNELS PRESENT
IN IF THEY WERE LABELED WITH
K11, K29, K33, WHICH IS THE
MINOR SPECIES, THEY ACTUALLY GET
SIDE TRACKS TO THE PROTEIN
COMPLEXIOSOME TO BE DEGRADED.
IN ORDER FOR THE CHANNEL TO
ACTUALLY ADD ATTRACT IT TO THE
CELL SURFACE, IT NEEDS K48 WHICH
ANOTHER PROTEIN IS A DEGRADATION
SIGNAL AND IN THIS CASE, IS A
TRAFFICKING SIGNAL TO GET TO THE
CELL SURFACE, FOR REMOVAL OF THE
CHANNEL, YOU REQUIRE K63, AND IF
YOU REMOVE K63, YOU SCROLL DOWN
THE HYDROLYSIS OF THE CHANNEL,
AND SO FORTH, AND SO WHAT THIS
INDICATE THAD INDEED THE
POLYUBIQUITIN CHAINS OF
DIFFERENT LINKAGES HAVE PROFOUND
DIFFERENT ASPECTS OF THE CALCIUM
CHANNEL.
SO THIS IS WHAT PEOPLE IN THE
UBIQUITIN FIELD CALL THE
UBIQUITIN CODE.
AND 1 THING I WOULD LIKE TO
STRESS IS THAT THE UBIQUITIN IS
MADE UP OF A SYSTEM OF EDITORS
AND SO FORTH.
AND THATIA GOING TO BE DIFFERENT
IN DIFFERENT CELLS, AND SO WE
COULD -- FOR EXAMPLE, WE SHOWED
THAT IF WE OVEREXPRESS, A
PARTICULAR TYPE OF LIGASE, WE
CAN AFFAIRS TEAM LEADERRER THE
UBIQUITIN CODE IN OUR LINKAGE
SELECTIVE ENDUBS ARE ABLE TO
DISTINGUISH THAT.
IF WE DO THE SAME EXPERIMENTS IN
HARP, THE OUTCOME IS DEFINITE
THAN WHAT YOU SEE IN HEX CELLS
BECAUSE THE UBIQUITIN CODE IS
DIFFERENT.
SO I THINK IT WILL BE A VERY
INTERESTING APPROACH TO BEGIN TO
DECODE THE UBIQUITIN FOR
DIFFERENT PROTEINS INSIDE THE
CELL.
ALL RIGHT, SO THE LAST STORY I
WANT TO TALK ABOUT REALLY, SO
FAR THE 2 STORIES I'VE BEEN
TALKING ABOUT ARE TALKING ABOUT
TARGETED DEUBIQUITIN TO
STABILIZE THE PROTEINS OR TO
LOOK AT ROLES IN ION CHANNEL
TRAFFICKING.
THE NICE THING ABOUT THE
UBIQUITIN SYSTEMS IS THAT YOU
CAN DO THE OPPOSITE, YOU CAN
REMOVE CHANNELS BY ADDING
UBIQUITIN TO THEM.
SO WE'VE BEEN LOOKING AT THAT
ASPECT OF THIS STORY AS WELL,
AND FOR THIS, THE -- THE --
WHERE WE MADE THE MOST HEAD WAY
IS LOOKING AT MY FIRST LOVE WHEN
IT COMES TO ION CHANNELS THAT
ALLOW CALCIUM TO COME IN AND
EXCITABLE CELLS TO LINK ACTION
POTENTIALS TO BIOLOGICAL
RESPONSES, OKAY?
SO 1 OF THE MAJOR THINGS WE'VE
BEEN INTERESTED IN AND THESE ARE
VERY IMPORTANT AND THERAPEUTIC
TARGETS, L-TYPE CALCIUM CHANNELS
ARE WELL KNOWN HYPE EAR
TENSIVES, THE NEURONAL GET
CALCIUM CHANNELS THAT ARE
TARGETED FOR THINGS LIKE
NEUROPATH WICK PAIN, PARKINSON'S
DEC AND FORTH.
SO THE LIMITATIONS OF SMALL
MOLECULE DRUGS BECAUSE THEY GO
EVERYWHERE AND THIS CHANNEL IS
PREVALENT IS MANY DIFFERENT
TISSUES CAN YOU GET OFF TARGET
OFF TISSUE EFFECTS THAT LIMIT
THE THERAPEUTIC WINDOW AND ALSO
SOME OF THESE CHANNELS LOOK SO
CLOSE TO EACH OTHER IT'S BEEN
DIFFICULT TO FIND SMALL
MOLECULES TO DISTINGUISH THEM.
SO MY LAB'S BEEN VERY INTERESTED
IN MAKING GENETICALLY ENCODED
CALCIUM BLOCKING CHANNELS THAT
CAN ADDRESS THESE DEFICITS.
SO THE CHANNEL IS MADE UP OF A
[INDISCERNIBLE], BUT MANY OF
THESE ALSO HAVE AN AUXILLIARY
BETA SUBSUANT THAT IS REQUIRED
FOR FORMATION OF THE CHANNEL SO
TO MAKE THESE CHANNELS 1 OF THE
FIRST THING WE DID WAS TO MAKE
AN NANO BODY TO THISAUK
ILLEGALSIARY BETA SUBUNIT.
AND WE DID THIS THE OLD
FASHIONED WEIGH, WE PURIFIED A
SUBBETTA UNIT, SENT IT TO A FARM
IN MASSACHUSETTS, THEY INJECTED
IT INTO A LLAMA AND THEY SENT US
THE BLOOD AND WE USED A PHASED
APPROACH TO FISH OUT THE
BINDERS.
SO HERE'S A BINDER WE FISHED OUT
F3, AND THIS INDICATES IT BINDS
ALL 4 TYPES OF BETA ISOFORMS
THAT ARE OUT THERE. SO WE HAVE
THESE NANO BODY BINDERS, WHAT DO
WE DO WITH THEM, WELL WE WILL DO
WHAT WE DID WITH THE ENDUBS IN
REVERSE.
THIS IN CASE, WE ARE LOOKING AT
AN E3 LIGASE, AND IT'S A MODULAR
PROTEIN AND HAS THESE W
W-DOMAINS THAT ALLOW IT TO BIND
TO THE CELL.
HAS A C2 DOMAIN THAT ATTACHES
TOLET MEMBRANE, BUT THE BUSINESS
END IS THE HECTODOMAIN WHICH IS
THE ENZYMATIC E3 LIGASE.
SO WE HIJACKED THE ENZYMATIC
PARTS, GET RID OF ALL THE OTHER
PARTS AND FUSE IT TO OUR NANO
BODY.
SO IN THIS WAY, IF WE EXPRESS
THE TAG NANO BODY WITH A CHANNEL
COMPLEX, MADE UP OF THE COMPLEX,
THE IDEA IS THAT THE NANO BODY
WOULD FIND A SUBUNIT AND CAUSE A
LOCALLAL UBIQUITIN OF THE
CHANNEL AND WE PREDICT THAD
WOULD CAUSE THE CHANNEL TO BE
DETAINED AND DEGRADED AS WELL.
SO WE WILL USE THE SAME FLO
CYTOMETRY ASSAY TO GET THESE TO
WORK IN THE CALCIUM CHANNELS.
SO WHEN WE EXPRESS THE CHANNEL
WITH JUST THE NAKED NANO BODY
FOR OUR CONTROL, YOU SEE A LOT
OF REG SIGNALS OF THE CELL
SURFACE IN YFP POSITIVE CELLS,
IF YOU ARM THE NANO BODY WITH A
HEX DOMAIN FROM THAT, THAT
SIGNAL IS ERASED.
INTERESTINGLY YOU CAN SEE THAT
THE PROTEIN IS DEGREATED, IT
DOESN'T GET TO THE CELL SURFACE,
AND THIS IS REALLY DEPENDENT ON
INFORMATION FWE MAKE IT
CATALYTICALLY DEAD VERSION OF
THE HEX DOMAIN, THE CHANNELS ARE
AT THE CELL SURFACE.
AS YOU EXPECT FROM THE
CHANNELSOT CELL SURFACE, MEASURE
THE CURRENT.
THIS IS 1 OF THESE IMPORTANT
CALCIUM CHANNEL PROHIBITOR,
PROFOUNDLY MOVES ALL THE OTHER
CURRENT.
THERE ARE MULTIPLE ISOFORMS OF
THESE FOR THE CALCIUM CHANNELS
THAT ARE IN THE BETA SUBUNIT,
AND THIS MOLECULE WAS VERY
EFFECTIVE FOR ALL OF THEM.
WE CALLED THEM CAPPA BLAITA AND
THIS IS WHAT TRAVIS IMAGINED IN
THE LLAMA WITH THE TERMINATOR.
SO WHAT IS IT GOOD FOR, 1 AREA
WE THOUGHT MIGHT BE USEFUL FOR
IS NEUROPATHIC PAIN CAUSED BY
LESIONS FROM THE NERVOUS SYSTEM,
DIABETES, NERVE INJURY, CHEMO
THERAPY AND SO FORTH, AFFECTS
MILLIONS OF PEOPLE.
IT'S A REAL MEDICAL NEED BECAUSE
IN FIRST AND SECOND LINE
MEDICATIONS ARE OFTEN INEECTIVE
OR HAVE VERY SERIES SIDE
EFFECTS, THE REASON WHY WE THINK
IT COULD BE USEFUL HERE IS
CONCENTRATION FROM THE CIRCUITRY
FOR SOPHISTICATED MAT O SENSORY
PAIN.
AND THAT SIGNAL HAS TO BE
TRANSFERRED, TO THE CENTRAL
NERVOUS SYSTEM WHERE YOU CAN
DECODE IT AS PAIN.
THE CENTRAL THING HERE IS THAT
THAT TRANSFER OF SIGNAL IS
DEPENDENT ON THESE CALCIUM
CHANNELS SO THE IDEA IS IF WE
COULD EXPRESS THAT FROM
HAPPENING HERE WE COULD PREVENT
IT FROM TAKING ROOT.
SO IF YOU EXPRESS THE NEURONS IT
COULD WEAPON OUT THE CURRENT.
SO WITH THAT IN HAND, THEN WE
LINKED UP WITH MY COLLEAGUE WHO
STUDIES ANIMAL MODELS OF PAIN
AND HERE'S THE EXPERIMENTAL
PARADIGM.
TWENTY-EIGHT DAYS BEFORE NERVE
INJURY WE CAN INJECT INTO THE
HIND PAW OF A MOUSE EITHER A
CONTROL AAV, 9 EXPRESSING
TDTOMATO, OR 1 THAT EXPRESSES
KAVABLATOR, AND THEN LOOK AT
PAIN.
SO 3 DAYS AFTER THE INJURY MODEL
THESE ANIMALS DEVELOP MECHANICAL
ALOE DEANIA WHICH YOU SEE AS A
DECREASE IN THE PAW WITH THE
THRESHOLD AND RESPONSE TO A
STIMULUS THAT WAS INITIALLY
[INDISCERNIBLE].
WHAT WE WERE GRATIFIED TO SEE
WAS THE ANIMAL THAT HAD
KAVATBLATOR EXPRESSED LESS PAIN
AND THAT PERSISTENT, WE'VE GONE
UP TO 2 YEARS, AFTER INYEKS AND
SEEN THAT THE CAVABLATOR IS
EXPRESSED, SO WE COULD GET THIS
RIGHT, DO A SINGLE INYEKS AND --
INJECTION AND GET YEARS OF PAIN
RELIEF WITHOUT ANY SED EFFECTS
AND THE EXTENT OF CAVABLATOR,
INCLUDES NASAA.COMMIA, HEAT AND
COLD.
SO AFTER WE PUBLISHED THIS,
[INDISCERNIBLE] AND HIS
COLLEAGUE PUBLISHED THIS IN THE
SPOTLIGHT IN TIPS, THE CALCIUM
CHANNEL TERMINATOR H
ASTA LA VISTA PAIN.
IF YOU LOOK AT IT, EVEN THOUGH
WE HAD LESS PAIN THAN THE
CONTROL, WE WERE STILL PROVING
SOME PAIN RELATIVE TO THE
WILD-TYPE CONTROL HERE SO THE
QUESTION WE HAD WAS WHETHER WE
COULD PUSH THIS UP TO CAUSE, YOU
KNOW LESS PAIN WITHOUT ANYTHING
HAPPENING AND 1 THING THAT A
PHARMACOLOGIST WILL TELL YOU IS
YEAH, JUST INJECT MORE DRUG BUT
GIVEN WHAT CAVABLATOR DOES WE
WERE WORRIED ABOUT IT SPILLING
OVER INTO OTHER ORGANS
ESPECIALLY THE HEART.
SO THE HEART IF YOU RECORD
CURRENTS, THE INWARD CURRENT
UNDER CONTROL CONDITIONS IS VERY
ESSENTIAL FOR YOU TO HAVE A
HEART BEAT THIS, IS WHAT
CAVABLATOR DOES WHEN IT GETS
INTO THE HEART, WE ARE VERY
AWARE THAT IF WE KEEP INJECTED
THIS WE WOULD BE SAYING
HASTA LA VISTA, POETIC THE
PATIENT.
SO HOW DO WE DO THIS WITHOUT
KILLING THE PATIENT?
SO 1 OF THE THINGS IS THAT THE
DIFFERENT BETA ISOFORMS OF THE
CHANNEL HAVE DIFFERENT TISSUE
EXPRESSION.
SO FOR EXAMPLE, THE HEART
EXPRESSES MOSTLY BETA 2, RIGHT?
AND O IF YOU COULD MAKE OTHER
KAVABLATOR ISOFORMS THAT COULD
BE AN INHIBITOR POTENTIALLY TO
GET TAKEN--THEY RELIEVE.
SO TRAVIS SCREENED THE SAMPLE
FROM THE LLAMA AND WE FOUND THAT
THE ISOFORM BAND AND NONE THE
OTHER BETAS AND WE COULD CAN IF
WE COULD MAKE A INHIBITOR BASED
ON THAT.
INDEED IF YOU FUSE THE DOMAIN TO
THE NANO BODY, YOU GET CHANNELS
THAT CONSITUTE THE BETA 1 AND
COMPLETE ELIMINATION OF THE
CURRENT AND THERE WERE TOTALLY
INERT AGAINST OTHER ISOFORMS OF
THE BETA SUBUNIT.
SO THERE'S A REAL NEW PARADIGM
FOR INHIBITING THE ION CHANNEL
BASED ON THE ISOFORM SUBTYPE OF
THE AUXILLARY SUBUNIT.
THE OTHER THING WE WERE WORRIED
ABOUT WITH THE CAVABLATOR, IF
YOU INJECT TOO, EVEN IF THE
EXPRESS ENOUGH OF IT WILL HAVE
OFF TARGET CENTER FOR EXCELLENCE
ON AGINGS SO WE WERE THINKING OF
WAYS TO AVOID.
A AND WE TOOK INSPIRATION FROM
THE PROTAX FIELD, AND PROTAX ARE
BINDERS, SMALL MOLECULES THAT
HAVE 1 PART THAT BINDS A LIGAND
AND ANOTHER PART THAT BINDS AN
E3 LIGASE, AND THE IDEA THAT IF
YOU GET INSIDE THE CELL IT
RECRUITS THE LIGASE TARGET AND
GETS DEGRADED AND WONDERED IF WE
COULD DO THE SAME THING WITH THE
NONVALENTINEDDENT NANO BODIES WE
CALL DIVAS, AND NOW GET THROUGH
TO BE RECRUITED.
THE ADVANTAGE OF A SMALL
MOLECULE OF COURSE IS THAT NOW
YOU CAN EXPRESS THIS IN
PARTICULAR CELLS THAT YOU ARE
INTERESTED IN INHIBITING IN THE,
SO IN ORDER TO DO THAT, THE
FIRST QUESTION WE HAD TO ADDRESS
WAS, YOU KNOW WHAT IS THE BEST
E3 LIGASE TO TARGET FOR ION
CHANNELS AND LOOKING AT THE
LITERATURE IT SEEMS LIKE NET
FORAND TO REALLY WELL KNOWN TO
REGULATE MULTIPLE ION CHANNELS.
SO THE QUESTION WAS WHETHER WE
COULD MAKE A RECRUIT NET 4 AND
NET 42, WHICH HAVEN'T BEEN USED
IN THE PROTAX FIELD TO INHIBIT
ION CHANNELS.
SO WITH THAT WE RAISED NANO
BODIES USING THE LIBRARY,
AGAINST THE HEK 42 DOMAIN.
WE FOUND 1 MAN NANO BODY C11
HERE THAT BINDS THEAT42 HECHT
4-2 DOMAIN AND IT BINDS A FULL
LENGTH IN HECHT-4-2 AS WELL.
AND IT OBTAIN A YIE O EM
STRUCTURE WITH OUR COLLEAGUE
OLIVER CLARK AND THE STUDENT
[INDISCERNIBLE].
AND FOUND WHERE IT BINDS SO WE
COULD LOOK AT THE INTERFACE AND
WHAT WAS IMPORTANT ABOUT THIS IS
THAT WE WANTED A NANO BODY THAT
WOULD BIND THE HECHT DOMAIN
WITHOUT DISRUPTING ITS FUNCTION.
AND BASED ON WHERE IT'S BIND --
WHERE THE NANO BODY BIND, WE
COULD INFER THAT IT DID IRPT
FEAR WITH ANY OF THE OTHER PARTS
THAT WERE IMPORTANT FOR THE
CATALYTIC ACTIVITY THE ENZYME,
THE CATALYTIC CYSTINE SITE, THE
E2 BINDING SITE AS WELL AS
SOPHISTICATED CALLED CRUCIATE
LIGAMENT CIBOL WITTEN SITE THAT
WAS IMPORTANT FOR POLYUBIQUITIN
CHAIN INFORMATION.
ALL RIGHT, SO NOW THAT WE HAD A
NANO BODY HERE WE COULD THEN SET
UP THIS SAME EXPERIMENT.
WE HAD A CHANNEL AND YOU HAVE A
3 CONDITIONS EITHER NAKED NANO
BODY TO THE BETA SUBUNIT OR YOU
CAN OVEREXPRESS NET 4 TO SEE IF
THE CHANNEL IS REGULATE BIDE
THAT OR YOU COULD RECRUIT
ENDOGENOUS M4 TO THIS
DISCIPLINARY VALENTINEDENT NANO
BODY OR THIS DIVA, OKAY.
SO THE EXPERIMENT, SO, THE
WILD-TYPE, IF YOU OVEREXPRESS
IT, THE RED SHOWS IF YOU GET IT,
YOU GET ABOUT 60% INHIBITION OF
THE CURRENT AND JUST PUTTING IN
THE DIVA TO RECRUIT ENDOGENOUS
NET 4-2 HAS THE SAME FUNCTIONAL
EFFECT AS THAT SO THAT WAS
HIGHLY EFFECTIVE TO DO THAT.
WE MADE AN AAV AND PUT THAT INTO
THE NEURONS AND INDEED,
RECRUITING DISCIPLINARY
VALENTINEDENT NANO BODIES,
RECRUITING ENDOGENOUS E32, AND
ENDOGENOUS CHANNELS IN THE
NEURONS ALSO CAUSE ABOUT A 70%
INHIBITION OF THE CURRENT.
ALL RIGHT, SO I WILL SUMMARIZE
WHAT I TOLD YOU THIS AFTERNOON,
I SPOKE ABOUT THE FIRST PART, AN
INCUB APPROACH, WAY TO RESCUE
AND MAKE MORE ION CHANNELS OR
ANY OTHER PROTEIN USING THE
TARGETED DECIBOL WITTENATION
STRATEGY, I SPOKE TO YOU ABOUT
THE CHALSIUM CHANNEL INHIBITOR,
THAT WE INITIALLY USED TO
INDICATE COULD BE POTENTIALLY
USED FOR LONG LASTING RELIEF OF
PAIN.
THE CHISELS ALLOW YOU MORE
ISOFORM SELECTIVITY AND SO THEY
DON'T HOPEFULLY KILL ANYBODY,
AND THEN THE DIVAS WE THINK CAN
BE A VERY GENERALIZED APPROACH
TO MAKING INHIBITORS FOR ANY ION
CHANNEL OR FOR OTHER PROTEINS AS
WELL.
SO THE FINAL THING IS I HAVE A
CONFESSION TO MAKE, I DIDN'T DO
ANY OF THIS WORK MYSELF, DONE BY
TALENTED GROUP OF STUDENTS AND
POST DOCS.
I MENTIONED THEM ALONG THE WAY
AND CERTAINLY 1 OF THE BEST
PARTS OF BEING A PROFESSOR AND
WORKING WITH REALLY SMART AND
TALENTED PEOPLE.
I HAVE A NUMBER OF WONDERFUL
COLLABORATORS AND ALSO, FINANCE
ALSO SO THANK YOU FOR LISTENING.
READY TO TAKE QUESTIONS.
[ APPLAUSE ]
>> THANK YOU FOR THE TALK.
QUESTIONS?
I WILL GET STARTED SO IN YOUR
INTRODUCTION, YOU TALKED ABOUT
LONG QT AND CFTR AND EPILEPSY,
DIDN'T LEE TALK ABOUT EPILEPSY
IN THE TALK, I'M INTERESTED, I
MEAN JUST THERE'S SO POTENTIALLY
CELL TYPE SPECIFICITY IN,A
DITION TO SUBTYPE, HAVE YOU
THOUGHT ABOUT ADDING CELL TYPE
INTO THIS?
AND HOW YOU COULD ADDRESS THAT?
>> SO, YEAH, 1 OF OUR -- A
COUPLE OF OUR PROJECTS ARE
DEALING WITH EPILEPSY AND
[INDISCERNIBLE] FOR EXAMPLE, IT
HAPLOSUFFICIENCY THAT OCCURS IN
NEURONS SO WE'RE APPLYING THIS
APPROACH TO ENHANCE THE EXPRESS
OF THE SODIUM CHANNELS IN THE
NEURONS FOR EXAMPLE, AND I THINK
THE -- HAVING A GENETICALLY
ENCODED APPROACH ALLOWS TO YOU
DO THAT SO CAN YOU DO IT IN A
SPECIFIC WAY YOU WANT TO.
>> [INAUDIBLE QUESTION FROM
AUDIENCE ]
>> REALLY GREAT QUESTION, SO
WHEN WE FIRST STARTED THIS
APPROACH, ALL I KNEW ABOUT
UBIQUITIN IS THAT YOU, YOU KNOW
IT DEGRADES THINGS SO WE WERE
LOOKING FOR DEGRADATION, AND WE
DID IT AT FIRST FOR A POTASSIUM
CHANNEL AND ALSO FOR THE CALCIUM
CHANNEL SO OUR FIRST WE WERE
JUST LOOKING FOR THE TOTAL
AMOUNT OF PROTEIN, DIDN'T
CHANGE.
HOWEVER, WHEN YOU LOOK AT THE
AMOUNT OF BURDEN ON THE CELL
SURFACE THAT WAS TOTALLY GONE,
SO IT TURNS OUT THAT WHAT THIS
WAS DOING IS CAUSING THE
CHANNELS TO BE TRAPPED INSIDE
THE CELL, RATHER THAN DEGRADING
THEM, SO IT WAS JUST NOT
UBIQUITIN PER SE, THAT'S
IMPORTANT AND I THINK THE SECOND
PART OF THE STORY WAS TELLING
THAT, RIGHT IN BECAUSE IT ALSO
REGULATES DIFFERENT ASPECTS OF
TRAFFICKING AS WELL, SO WE'RE
INTERESTED IN LOOKING AT THESE
SIGNALS THAT REGULATE EVERYTHING
FROM THE TRAFFICKING PATHWAY TO
WHAT ACTIVATES RESPONSIBLE FOR
DEGRADATION, WE THINK IT WILL BE
DIFFERENT TYPES OF UBIQUITIN
CHAINS AND SO FORTH.
>> SO A QUICK FOLLOW UP
[INDISCERNIBLE] SURFACE AND
APPROXIMATELY CHANNELIZE
TARGETING [INDISCERNIBLE]?
>> I THINK IT'S A COMBINATION OF
THE 2.
SO FOR THE SECOND STORY, FOR
EXAMPLE, WHERE WE HAD A NANO
BODY TIED TO THE YFP, SO IT'S
ALWAYS GOING TO BE THE CYTOSOLIC
PART AND YOU LOOK AT THE RATE OF
FORWARD TRAFFICKING VERSUS RATE
OF -- YOU EFFECT BOTH THOSE
PROCESSES SO WE THINK YOU ARE
AFFECTING EVERYTHING IN THAT
SENSE.
>> QUESTIONS?
>> [INAUDIBLE QUESTION FROM
AUDIENCE ]
>> K63 PREDOMINANTLY.
MAKES, SENSE.
RIGHT.
WE DID SOME -- WE ALSO HAVE A
NUMBER OF LIGASES AND SOME ARE
ABLE TO DEGRADE MORE THAN
OTHERS.
THAT'S CONSISTENT.
>> [INAUDIBLE QUESTION FROM
AUDIENCE ]
>> THAT'S RIGHT, THAT'S RIGHT.
YEAH.
>> YEAH, SO, FOR SOME OF YOUR
RESIDENT EXPERIMENTS ESPECIALLY
FOR LQTS, THERE WAS AN F508
DELETION THAT YOU HAD, DID YOU
DO ANY PHYSIOLOGICAL ASSAYS TO
DETERMINE THE EFFECT OF THE PACE
ON THE HEART AFTER THOSE RESCUE
EXPERIMENTS, BUT I KNOW YOU HAD
A RESCUE EFFECT BE, WAS THERE
ANY EFFECT OF TACHYCARDIA OR
SOMETHING OF THAT NATURE?
>> YEAH, SO THE F5, I THINK THAT
WAS CFTR, THAT WAS THE CHLORIDE
CHANNEL SO THAT WAS THE CASE OF
THE CYSTIC FIBROSIS.
SO FOR THAT PARTICULAR CASE, I
THINK THE SUPER RESCUE WOULD
PROBABLY BE FINE BECAUSE, YOU
KNOW YOU JUST GET MORE HYDRATION
OF THE AIR WAYS BUT YOU KNOW
THERE ARE CERTAIN
CONSIDERATIONS, IF YOU -- THE
GENERAL QUESTION IS, CAN YOU
OVERRESCUE SOMETHING, RIGHT IN
WHAT WE FOUND IN GENERAL IS THAT
IT'S VERY HARD TO GET MORE THAN
2-3 FOLD INCREASE IN ANYTHING,
AND JUST LIKE ANY OTHER DRUG,
YOU WOULD HAVE TO TITRATE IT TO
A THERAPEUTIC SORT OF LEVEL.
THANK YOU.
>> BEAUTIFUL TALK.
ACTUALLY A QUESTION ABOUT THE
SAME PHYSIOLOGICAL THINGS YOU
HAD SHOWN BEFORE, IT WAS
INTERESTING THAT YOU SHOWED IT
WAS NOT ONLY A TRAFFICKING
DEFICIT BUT ALSO A DEFICIT IN
THE YOU KNOW TOTAL
[INDISCERNIBLE] REACHED THE
SURFACE AND SO, I GUESS WHEN I
THINK OF UBIQUITINNATION, I
THINK OF PROTECTING AGAINST
DEFECTIVE OR NONFUNCTIONING,
CHANNELS THAT DON'T FUNCTION
PROPERLY, DO YOU THINK YOU WOULD
BE ABLE TO SORT OF MAINTAIN THE
INTEGRITY OF THE CHANNEL THAT
MAKE ITS TO THE SURFACE IN
ADDITION TO INCREASING, YOU KNOW
BEYOND JUST INCREASING SURFACE
EXPRESSION, MAKING SURE THAT THE
1S THAT ARE FUNCTIONAL, IS THAT
SOMETHING WITH NANO BODY
TECHNOLOGY COULD DO?
>> YEAH, SO, MAYBE I'LL TAKE AN
EXAMPLE FROM THE
CYSTIC FIBROSIS, THE VERTEX
DRUG, IT'S A TRIPLE COMBINATION
THERAPY, RIGHT?
TWO OF THE DRUGS HELP THE
CHANNEL FOLD BETTER IN TRAFFIC
AND THIS IS SOMETHING WE FOUND
AS WELL.
FOR THE CYSTIC FIBROSIS CHANNEL,
WE JUST ADD THE ENDUB, WE CAN
RESCUE THE CURRENT, THE SURFACE
DENSITY BY ABOUT A HUNDRED
PERCENT BUT WHEN YOU MEASURE THE
CURRENT, IT'S TERRIBLE, STILL SO
BAD, THAT'S WHAT THE MUTATION
DOES IT AFFECTS THE ABILITY OF
THE CHANNEL TO FOLD PROPER LE.
SO 1 OF THE DRUGS THIS HAS BEEN
SHOWN IN USING STRUCTURAL
BIOLOGY, IS THAT THE DRUG
ACTUALLY HELPS THE CHANNEL TO
FOLD BETTER AND SO, FOR THE
CYSTIC FIBROSIS, WE NEEDED THE
MAUL MOLECULE DRUG IN,A DITION
TO THE ENDUB TO GET THE MAXIMUM
RESCUE, RIGHT?
AND SO, IT'LL BE DEFINITE FOR
DIFFERENT MUTATIONS AND WHAT
THEY DO.
>> IF THAT'S EVERYTHING, THEN
LET'S THANK HENRY AGAIN.
[ APPLAUSE ]