>>WELCOME TO THE WEDNESDAY
AFTERNOON LECTURE SERIES. THIS
IS OF COURSE THE NIH-WIDE FORUM
FOR THE MOST DISTINGUISHED
SCIENTISTST IN BIOMEDICAL
RESEARCH. I'LL BE HOSTING THE
SPEAKER TODAY. HAU WU FROM THE
FIELD OF STRUCTURAL BIOLOGY AND
VISITING FROM HARVARD MEDICAL SCHOOL.
DR. HAO WU IS A PROFESSOR
AT HARVARD AND ALSO AN ASSOCIATE
DIRECTOR AT THE BOSTON
CHILDREN'S HOSPITAL AND WORLD
RENOWNED FOR HER WORK ON SIGNAL
INSTRUCTION IN CELL DEATH. SHE
EARNED HER BACHELOR'S DEATH IN
BEIJING, CHINA AND THEN WENT TO
PEKING UNION MEDICAL COLLEGE,
WHILE AT COLLEGE SHE
PARTICIPATED IN LABORATORY
RESEARCH AND IMMUNOLOGY AND AS
RUMOR WOULD HAVE IT
WITH RESEARCH AND
RECEIVED A PH.D. FROM PURDUE
UNIVERSITY. HER POSTDOC WAS AT
COLUMBIA UNIVERSITY WITH ANOTHER
RENOWNED STRUCTURAL BIOLOGIST
WAYNE HENDRICKSON AND IN 1997
BECAME A FACULTY MEMBER AT THE
COLLEGE OF MEDICINE IN NEW YORK
CITY. SHE WAS THERE FOR FIFTEEN
YEARS AND MOVED HER LAB
NORTHWARD TO HARVARD. AMONG HER
MANY, MANY LANDMARK DISCOVERS
AND I CAN SAY THIS FROM PERSONAL
EXPERIENCE BECAUSE WE HAD THE
GOOD FORTUNE TO COLLABORATE WITH
HER. WAS IDENTIFYING THE
ASSEMBLIES OF VARIOUS PROTEINS
WITHIN THE CELL THAT CARRY OUT
IMPORTANT FUNCTIONS IN CELL
DEATH AND IMMUNITY.
PARTICULARLY THE PROTEINS THAT
WE HAD A STRONG INTEREST IN FROM
A PUBLIC HEALTH AND DISEASE
STANDPOINT I WOULD SAY SHE
REWROTE THE TEST BOOKS WHEN SHE
DISCOVERED THE INFLAM MA SOEM
ACTIVATION THROUGH THESE HIGHER
ORDER ASSEMBLIES.
A THEME THAT GOES ON THROUGHOUT
HER WORK AND ON MANY DIFFERENT
IMPORTANT ASPECTS OF THE IMMUNE
SYSTEM. SHE FOUND THAT THESE
DEBT DOMAIN COMPLEXES HAVE A
HELIO SYMMETRY THAT HARKENS BACK
TO WORK BY FRANCIS AND OTHER
OUTSTANDING BIOLOGISTS AND HAVE
UNUSUAL STOIC I DON'T MEAN
TRIES. 5-7 FOR THE PIDD, RAIDD
COMPLEX FOR BASE TWO ACTIVATION.
AND IN THE CAS BAIT 8
ACTIVATION. THE TNF REACCEPT
FORFAMILY AND 644 MORE MID 8.
THESE ARE VERY UNUSUAL
STRUCTURES I DON'T KNOW IF THOSE
ARE FIBONACCI NUMBERS OR WHY
NATURE EVOLVED TO HAVE THOSE
NUMBERS BUT CAME DIRECTLY FROM
HER INSIGHTS. HER LAB HAS DONE
OTHER THINGS. THE HALLMARK OF
ALZHEIMER'S DISEASE AND HAS
BECOME AN OUTSTANDING CRY OWE
EM. SCIENTIST IN ADDITION TO
HER WORK IN CRYSTALLOGRAPHY SO
NOW SHE'S IMAGING EVEN LARGER
STRUCTURES IN THE CELL AND I
JUST WANT TO POINT OUT THIS IS
JUST A SNAPSHOT OF HER
REMARKABLE CAREER. SHE'S HAD
MANY, MANY HONORS FOR THE
CONTRIBUTIONS SHE'S MADE. SHE
WAS RECIPIENT OF THE MAYOR'S
AWARD FROM NEW YORK CITY AND I
ASSUME YOU GOT THAT FROM MIKE
BLOOM B
BLOOMBERG. THIS YEAR SHE WON AN
AWARD FROM THE ASSOCIATION FROM
IMMUNOLOGISTS AND ALSO ELECTED
FOR THE ADVANCEMENT OF SCIENCES
AND THE NATIONAL ACADEMY OF
SCIENCES AND FROM US RECEIVED
THE PRESTIGIOUS PIONEER AWARD
GRANT WHICH IS GIVEN TO VERY FEW
OF THE OUTSTANDING SCIENTISTS IN
THE UNITED STATES AND IT MUST
MAKE HER FEEL VERY GOOD TO BE AN
HONOR PROFESSOR IN PEKING UNION
MEDICAL COLLEGE, HER ALMA MATER
AND AS WELL AN AN HONORARY
PROFESSOR. THANK YOU FOR BEING
HERE WITH US TODAY. AND I
WELCOME YOU TO NIH.
>> (APPLAUSE).
>> THANK YOU SO MUCH MIKE, THAT
WAS VERY KIND OF YOU.
I'M DELIGHTED
TO BE HERE. ACTUALLY I HAD GREAT
FUN TALKING TO SOME OF YOU
TODAY. THIS MORNING WITH PIS AS
WELL AS STUDENTS AND POSTDOCS.
SO I WOULD LIKE TO SPEND THE
TIME TODAY TO TELL YOU SOMETHING
THAT WE HAVE BEEN WORKING ON FOR
A LONG TIME BUT NOW JUST FINALLY
THEY ARE COMING TOGETHER AS WELL
AS SOME UNPUBLISHED DATA. AND
GUESS WHO I BUMP INTO THIS
MORNING? TRYING TO GET
BREAKFAST. SO HERE ARE MY
DISCLOSURES. SO LET ME GET
RIGHT TO IT. SO INFLAMMASOMES
WERE FIRST IDENTIFIED, JUST WANT
TO MAKE SURE THIS MOUSE WORKS.
YEAH, OH. IS THIS THE PEN?
OKAY. IDENTIFIED AS THE SUPER
MOLECULAR COMPLEX THAT ACTIVATE
ONE AND THIS CAME FROM YOURJURG
LAB. THIS IS A PC, IT'S NOT A
MAC, OKAY. THE FIRST INFLAM
SOME WAS ACTUALLY IDENTIFIED IN
MY FRIEND AND COLLABORATOR'S
LAB. THE LATE JURG AS YOU CAN
SEE FROM THE BEGINNING IT IS
ALREADY KNOWN THAT INFLAM SOMES
CONTAIN A PROSEEN NOPE AS THE
SENSOR AND THEN THE ADAPT TOR
AND THEN OF COURSE CASPASE ONE
BECAUSE THAT'S THE FUNCTION FOR
THE INFLAMMASOMES AND THE FACT
THAT THEY FORM THESE LARGE KNOW
LEB YOU WHAT ARE ASSEMBLIES AS
YOU CAN SEE FROM THEIR MIGRATION
FROM THEIR COLUMN.
SO AS YOU CAN SEE, FROM THIS
TIMELINE, THE INFLAMMASOME
DISCOVERY REALLY WAS A
BREAKTHROUGH FOR THE FIELD OF
FEVER AND INFLAMMATION BECAUSE
IT FOLLOWS THE FIRST DISCOVERY
OF THE INTERLIEU ONE-1 CYTOKINE
AND THEN THE CASPASE-1 THAT
CLEAVES. SO NOW OF COURSE,
INFLAMMASOME ACTIVATE THIS
CASPASE-1. WE NOW KNOW THAT
INFLAMMASOMES ARE COMPOSED OF
THESE SUPERMOLECULAR STRUCTURES
AND I LIST HERE SOME OF THE
SENSOR PROTEINS THAT'S IN THESE
INFLAMMASOMES AND IN TODAY'S
TALK I WILL TALK ABOUT SOME OF
OUR WORK ON THE INFLAMMASOME, AT
LEAST THAT WILL BE PART OF THE
FOCUS OF THE TALK. SO THESE
SENSOR PROTEINS UPON ACTIVATION
BY PATHOGENIC OR ENDOGENOUS
DANGER SIGNALS, IT COULD ADAPT A
PROTEIN AND THEN CASPASE-1 OF
COURSE AND THIS WILL GENERATE
AUTO PROCESSING OF CASPASE-1 TO
INDUCE INFLAMMASOME ACTIVATION
SO THE INFLAMMASOME ITSELF OFTEN
FORMS AT A SINGLE SPEC IN THE
CELL. SO THIS I'LL COME BACK
LATER. THIS IS A SPECIAL
LOCATION IN THE CELL. AT THE
MICROTUBULE ORGANIZING CENTER SO
ACTIVATING CASPASE-1 OR OTHER
CASPASES THAT ARE ALSO
PROINFLAMMATORY WHICH ARE
INDUCED TO ACTIVATE THROUGH WHAT
WE CALL A NON CANONICAL
INFLAMMASOME PATHWAY CAN ALL
CLEAVE THIS PROTEIN KNOWN AT GAS
DWER PIN D INITIALLY DISCOVERED
IN THE LAB. FROM ONE DAY USED
GENETIC APPROACHES TO TRY TO
LOOK FOR PROTEINS INVOLVED IN
PAR RIP TO SIS. SO IT TURNS OUT
IT CAN BE CLEAVED BY THESE
CASPASES AND THEN THE
INDETERMINABLE REGION THAT'S
RELEASED FROM THIS CLEAVAGE AS
WE SHOWED AND SEVERAL OTHER LABS
SHOWED INTERACT WITH LIPIDS AT
THE CELL MEMBRANE AND THEN FORM
PORES AND THESE PORES ARE
IMPORTANT FOR THE RELEASE OF THE
MATERIAL CYTOKINE TO THE OUTSIDE
OF THE CELL BECAUSE THESE MATURE
CYTOKINES DO NOT HAVE A SIGNAL
SEQUENCE AS WELL TO INDUCE THE
CELL DEATH THAT'S HIGHLY
INFLAMMATORY KNOWN AS PIE ROP TO
SIS. SO LET ME START FROM THE
NRP 3 INFLAMMASOME. A LITTLE
BIT DIFFERENT FROM OTHER IMMUNE
SENSORS. NRP 3 IS NOT A
RECOGNITION RECEPTOR. INSTEAD
IT SENSES SOMETHING MORE GENERAL
AND THE EMERGING CONCEPT IS THAT
NRP 3 SENSES SOME KIND OF A CELL
MEMBRANE DAMAGE. SO MANY
DIFFERENT THINGS CAN ACTIVATE
NLRP 3. I HIGHLIGHT A COUPLE
HERE.
ONE IS THE BACTERIAL TOXIN NIGER
RICIN BECAUSE IT ENDANGERS THE
CELL MEMBRANE OR EXTRACELLULAR
ATP WHICH IS A SIGNAL THAT CAN
ACTIVATE A CHANNEL. AND THIS
PARTICULAR STRUCTURES IN A CELL
THAT CAN EACH INDUCE OR
ASSOCIATE IT WITH CERTAIN
IMPORTANT HUMAN DISEASES SUCH AS
YOUR RISK ACID FOR GOUT.
CHOLESTEROL CRYSTALS AND EVEN MP
2.5 FOR ENVIRONMENTAL LAWN
DISEASES. SO NLRP 3 IF YOU LOOK
AT ITS DOMAIN OPTIMIZATION HAS
THIS POWER DOMAIN AND THIS
INDETERMINATES IS PART OF THE
DOMAIN FAMILY AND THEN IT HAS
THE SO-CALLED DOMAIN THAT IS
ESSENTIALLY ATPASE BUT IT'S
FUNCTION IS NOT TO GENERATE A
FORCE, FOR EXAMPLE, DUE TO APT
HYDROLYSIS BUT RATHER, BY
BINDING TO ATP SOMEHOW INDUCES
AGGREGATION OF THE PROTEIN IN
ORDER TO PERFORM ITS FUNCTION
AND THE DOMAIN IS WELL-KNOWN FOR
THE FUNCTION. THE AC ADAPTER
ALSO HAS A PYD PROTEIN AND THEY
CAN INTERACT THROUGH THESE
STRUCTURES THAT IS COMMON TO THE
ENTIRE DOMAIN FAMILY. CARD IS
ALSO A FAMILY MEMBER. AND ALSO
ABLE TO RECRUIT THE CASPASE
MOLECULES WHICH THEN BRING THE
CASPASE TOGETHER FOR THEIR
ACTIVATION. SO INTERESTINGLY IN
CELLS, THEY FORM THESE LARGE
SPECS, THAT ARE ESSENTIALLY
LARGE AGGREGATES THAT CAN
FURTHER KA CAPITULATE THEM IN VO
SO THE QUESTION IS HOW DO THEN
NLRP 3 FORM THE INITIAL PLATFORM
IN ORDER TO KNEW COLLIUATE THE
FORMATION OF THE PYD AND THE CA
KRS
CARD AND I WILL GO THROUGH THE
JOURNEY THAT WE WENT THROUGH
THROUGH THREE DIFFERENT
STRUCTURES AND THIS EXTENSIVE
INTRACELLULAR TRACKING THAT WE
DISCOVERED ALONG THE WAY SO THE
FIRST STRUCTURE THAT WE SOLVED
WITH NLRP 3 IS THE NLPR 3 THAT
DOESN'T HAVE THE PYD. WE WORRY
THAT HAD THE ABILITY WOULD MAKE
THIS PROTEIN HIGHLY AGGREGATE.
SO WE SOLVED THIS STRUCTURE IN
COMPLEX WITH NEK 7. WHAT IS NEK
7? NEK 7 IN FACT IS A CENTRO
SEW MALL KINASE. AND IT THE
CATALYTIC ACTIVITY IS NOT
REQUIRED FOR ITS SUPPORTING OF
NRLP 3 ACTIVATION BUT THE
SCAFFOLDING ACTIVATION THAT IS
REQUIRED.
WHEN WE SAW THE STRUCTURE WE SAW
THE DOMAIN, DIRECT LY KO THIS NK
7. WE ALSO KNOW THE NLRP 3 IS
IN THE ADP BOUND STATE WHICH IS
INACTIVE SO THEN A COUPLE YEARS
AGO, WE SOLVED THE STRUCTURE OF
THE RP 3 WITH THE PYD EVEN
THOUGH THE PYD IS THERE IT LOOKS
LIKE IT'S COMPLETELY BURIED
WITHIN THIS -- WHAT WE CALL A
DOUBLE RING CAPABLE. A CAPABLE
THAT HAS TWO LAYERS. 506 ON TOP
AND 506 ON THE BOTTOM AND PYD IS
HIDDEN INSIDE THIS CAGE AND
THAT'S PROBABLY WHY THAT THE
NLRP 3 IS PROBABLY STILL
INACTIVE AND THE NACHT DOMAIN IS
STILL ADP BOUND NOT ATP BOUND.
WHAT IS SURPRISING IS WHEN WE
LOOKED AT THESE CAGES WE FOUND
THEY'RE MOSTLY MEMBRANE BOUND SO
WHEN WE FRACTION NATE THESE
CELLS MOST OF THEM ARE IN THE
MEMBRANE FACTION AND IF WE SOL
YOU LOCALIZE THEM WE GET THE
SAME KIND OF CAGE-LIKE STRUCTURE
THAT WE SAW FROM THE SOLUBLE
FRACTION. IN FACT THIS CAN BE
CAPITULATED. IN THIS CASE,
HERE, FOR EXAMPLE, IN THE WILD
TYPE IMMORTALIZED BONE MARROW
DERI
DERIVED. THE FRANK HAS A BAND
THAT CORRESPOND TO THE PURR FIE.
WHERE THE PURIFIED WOULD HAVE TO
RUN ON A NATIVE PAGE.
SUGGESTING THESE MEMBRANES ARE
CONTAINED IN THE CAGE SO THIS
CAGE HAS THIS INTERESTING
STRUCTURE THAT THIS REGION HERE,
THIS REGION HERE, WHICH IS THE
LINKER BETWEEN THE PYD AND THE
MATCH DOMAIN RATE RIGHT HERE.
THIS REGION HERE HAS A LOT OF
POSITIVELY CHARGED RESIDUES.
AND WE SHOW THAT WHEN YOU MUTATE
THESE RESIDUES, THEY ACTUALLY
CAN NO LONGER BE ON THE MEMBRANE
AND THEY ALSO CANNOT FORM THE
CAGE SUGGESTING THAT THIS
BINDING TO THE MEMBRANE IS
IMPORTANT FOR CAGE FORMATION.
SO JAMES CHAN'S LAB A FEW YEARS
AGO FOUND THAT THE STIMULATION
INDUCES THIS. SO THE GOLGI
WHICH USUALLY IS A CAP LIKE
STRUCTURE RIGHT NEXT TO THE
NUCLEUS IMPOUND THE INFLAMMASOME
THE CAP COMPLETELY DISAAPPEAR
AND BECOME VESICLES AND THEY GET
TRAFFICKED TO THE MTOC. THIS IS
AT THE MTOC AND THIS IS A TGN
AND THESE ARE TRAFFICKED TO THE
MTOC TO BRING NLRP 3 TO THE MTO
KRVRJTS THIS IS A KINASE AND WE
WILL COME BACK TO THAT IN A BIT
THEN WE SHOW THE CAGE. WHY DO
WE HAVE TO STRUCTURE. IT'S KIND
OF A COMPLICATED STRUCTURE THAT
PREVENTS ACTIVATION BUT WHY DO
WE NEED IT FOR NLRP 3 ACTIVATION
SO IT TURNED OUT THE CAGE ITSELF
IS IMPORTANT FOR THE DISPERSION
OF THE TGN AFTER NLRP 3
STIMULATION. IN THE WILD TYPE
MACROPHAGES, THESE ARE THE TGN
STAINING. THESE ARE UPON
STIMYOU LAYING WITH THE
BACTERIAL TOXIN AND THE TGN THEY
ALL ARE HERE AND DISPERSE INTO
TINY VESICLES AND THIS
DISPERSION IS DEPENDENT. IF YOU
USE THE KNOCKOFF CELLS YOU DON'T
GET THIS DISPERSION UPON
TREATMENT WITHIN THE ACTIVATOR
AND IF YOU RECONSTITUTE THE
CELLS WITH A MUTANT OF NLRP 3
THAT CANNOT FORM THE CAGE YOU
ALSO DON'T GET TGN DISPERSION
SUGGESTING THAT THE CAGE IS
IMPORTANT FOR THIS PROCESS SO
THIS LED US TO HYPOTHESIS THAT
THE CAGE ITSELF MAKES THIS FOR A
REASON. IT NOT ONLY SUPPRESSES
THE NLRP 3 ACTIVATION BUT ALSO
IMPORTANT FOR THIS EARLY STEP IN
THIS ACTIVATION BY DISPERSING
THE TGN SO NOW WE KEPT ON
SOLVING NLRP 3 STRUCTURE IN THE
CONFIRMATION. NO MATTER WHAT WE
DO, EVERYTHING THAT WE PURIFY IS
IN THE INACTIVE CONFIRMATION SO
THIS CHANGED WHEN MY POSTDOC LE
DECIDED TO TRY SOMETHING ELSE.
HE COEXPRESSED NLRP 3 WITH NEK
7. AND THEN HE TREATED THE
CELLS WITH NIGH OWE RICIN,
BASICALLY TREAT THE CELL WITH
THE SAME STIMULUS AND THEN HE
ADD THE ATP ANALOG. TO THIS TO
STABILIZE THE CONFIRMATION AND
ON TOP OF IT HE ADDED ASC
BECAUSE IF WE HAVE -- IF WE
DON'T HAVE ALL THREE OF THEM WE
DO NOT GET THIS ORDERED
STRUCTURE. I THINK PARTLY
BECAUSE THE NLRP 3 INFLAMMASOME
UNLIKE THE STRUCTURES WHICH WE
SOLVED, IN THERE, ASC WAS NOT
REQUIRED FOR THE COMPLEX BUT
HERE YOU NEED THE ASC TO TABLIZE
THE NLRP 3 COMPLEX. YOU CAN
ONLY MAINTAIN IT BETWEEN NLRP 3,
NEK 7 ANDS ASC AND YOU CAN SEE
THE STRUCTURES THAT ARE EVEN 11
FOLD OR 10 FOLD AND THE
STRUCTURE WITH THE 10 FOLD REACH
THE BEST RESOLUTION AND
THEREFORE I'M SHOWING YOU THE 10
FOLD STRUCTURE. SO THIS IS THE
INDIVIDUAL SUBUNIT. IN THE MAP,
THIS IS WHERE N EK 7 IS. SORRY,
I'M NOT DRAWING IT WELL. THE
YELLOW IS NEK 7. EVERYTHING
ELSE IS NLRP 3. THIS WHOLE
THING HERE AND THIS DOMAIN NOW
HAS CHANGED THE CONFIRMATION TO
AN ATP BOUND CONFIRMATION AND IF
YOU LOOK AT THE SITE VIEW YOU
CAN SEE THAT THE ADDED CYPD IS
TOGETHER WITH THE -- NLPR 3 TO
FORM A SHORT FILAMENT NEXT TO
THE DISC. SO JUST SHOWING YOU
BRIEFLY THE CONFIRMATION CHANGE
THAT'S OCCURRING. BASICALLY
THIS DOMAIN AS WELL AS PART OF
THE ENACT DOMAIN TOGETHER HAVE
THIS MASSIVE MOVEMENT. ALMOST
90 DEGREE MOVEMENT AND I WON'T
DETAIL THESE OTHER CHANGES BUT
THERE'S ALSO LOCAL CHANGES IN
OTHER IMPORTANT DOMAINS IN THE
PROTEIN ESPECIALLY THIS DOMAIN
THAT HAS BEEN IMPLICATED AS A
SENSOR FOR INFLUX. I THINK
THERE'S A LOT MORE STORY TO THAT
BUT WE DON'T KNOW HOW THIS
DOMAIN OR OTHER ASPECTS OF THE
BIOLOGY SENSES THE ACTIVATORS SO
HERE I JUST SHOW YOU THAT
INSTEAD OF ATT -- ADP -- NOW WE
HAVE ATP BOUND AND THIS RESIDUE
HERE HISTIDINE 522 IN THE
INACTIVE CONFIRMATION WOULD HAVE
BEEN IN CLASH WITH THE ACTUAL
GROUP IN THE ATP AND THEREFORE,
THIS WOULD INDUCE A
CONFIRMATIONAL CHANGE TO THE
ACTIVE STATE WHEN ATP IS BOUND.
SO MOST PROMINENT QUESTION FROM
THE STRUCTURES I THINK IS THE
FACT THAT NEK 7 WHICH I SHOWED
YOU THIS BEFORE, ALREADY, NEK 7
WHICH IS RIGHT HERE. IT DOESN'T
REALLY ASSOCIATE WITH ANYTHING.
I DOESN'T ALLEVIATE THIS AND THE
QUESTION WOULD BE THEN WHAT IS
THE FUNCTION OF NEK 7? SO WE
HYPOTHESIZED BECAUSE NEK 7 ALSO
BINDS THE LEUCINE RETREAT DOMAIN
AND THIS IS THE SAME AS FOR THE
LEUCINE RICH DOMAIN INTERACTION
IN THE CAGE. SO WE HYPOTHESIZE
THAT THESE TWO BINDINGS ARE
COMPETING WITH EACH OTHER. AND
INDEED IF WE START WITH A CAGE
STRUCTURE AND THEN WE ADD NEK 7
AS SHOWN HERE, THE CAGE WHICH IS
THIS PEAK HERE AND THIS PEAK
HERE THE CAGE WOULD BE DISRUPTED
AND SUGGESTING THAT INDEED, NEK
7 CAN DISRUPT A CAGE SO THIS
BIOCHEMICAL DATA AS WELL AS CELL
BIOLOGY AND STRUCTURAL DATA
PROMPT US TO PROMOTE -- TO --
PROPOSE THIS HYPOTHESIS OF
TRAFFICKING AND CONFIRMATIONAL
CHANGES IN ACTIVATION. THAT IS,
STARTING THERE MONOER HERRIC
NLRP 3 OR THE CAGE FORM AT THE
WORK. THERE MAY BE AN
EQUILIBRIUM BETWEEN THIS STATE
AND THE CAGE STATE. APPARENTLY
IN NLRP 3 ACTIVATION WE IMAGINE
THAT THE ATP EXCHANGE WOULD HAVE
OCCURRED ALREADY SO THAT THIS
NEK DOMAIN CHANGES CON FIR MAKE.
THIS CLOSED CONFIRMATION IS MORE
COMPACT WHEREAS THE ACTIVE ATP
BOUND CONFIRMATION IS MORE
STANDING UP LIKE THIS AND THEN
THIS KIND OF CAGE IS ABLE TO BE
TRANSPORTED TO THE MTOC ON THE
MICROTUBULE NETWORK. THIS
ACTUALLY BEEN KNOWN FOR UNITED
STATES SOME TIME AND ONE OF THE
FIST DISCOVERIES OF
INFLAMMASOMES IS THAT IT IS
SENSITIVE TO MICROTUBULE
STRUCTURES AND IN FACT THIS IS
AN INFLAMMASOME DRUG. ONCE IT
GETS TRAFFICKED WE HYPOTHESIZE
NEK 7 WOULD BE THERE TO DISRUPT
THE CAGE AND BECAUSE THE TWO LAY
NEVERS -- LAYERS ARE ALREADY
LINKED TOGETHER. ONE CAN
IMAGINE THAT THIS WILL JUST
UNFURL. UNFURL THE CAGE INTO
TWO FIVE SUBUNIT SUB COMPLEX.
AND THEN THESE TWO CAN JUST COME
TOGETHER TO FORM THE FINAL DISC
THAT CONTAINS TEN SUBUNITS AND
THIS OF COURSE NEED TO BE
FURTHER STABILIZED BY THE BOUND
ASC SHOWN HERE IN RED. WHY IT
USE Z SUCH A COMPLEX ACTIVISM,
WE THINK ANY INFLAMMASOME
ACTIVITY. IF YOU PRODUCE DEATH
THAT'S THE END OF THE CELL SO
PROBABLY THE CELL HAVE DEVELOPED
MANY DIFFERENT REGULATORY
MECHANISM IN ORDER TO SUPPRESS
THIS ACTIVATION UNTIL EVERY STEP
IS CLICKED. EVERY STEP, EVERY
CHECKPOINT IS CLICKED. SO THAT
NLRP 3 CAN BE ACTIVATED. SO
INTERESTINGLY NLRP 3 IS
CONSERVED THROUGHOUT EVOLUTION
AND BAT IS AN INTERESTING CASE.
SO THE BAT IS KNOWN AS A
RESERVOIR FOR MANY DIFFERENT
VIRUSES AS YOU KNOW. WHICH MAY
INCLUDE THE CORONAVIRUS. THE
PANDEMIC THAT WE HAVE JUST
SUFFERED THROUGH. SO IT TURNED
OUT THEIR SAY SEW FORMS ARE
DIFFERENT AND THIS MEANS IT IS
DELETED.
IF YOU LOOK AT THE DELETED
ISOFORMS IT HAS -- WHICH IS
ISOFORM 2 AND 4 IT HAS VERY LOW
ACTIVITY AS IN 2 HERE AND 4
HERE. VERY LOW ACTIVITY SO WE
HYPOTHESIZED THAT BECAUSE OF
THIS DELETION IN THE DOMAIN, IT
STILL MAY BE ABLE TO FORM THE
CAGE BUT MAY NOT BE ABLE TO BIND
TO NEK 7 AND THEREFORE YOU
CANNOT ACTIVATE THE BAD NLRP 3
INFLAM SOME AND THIS IS
SOMETHING WE'RE STUDYING.
ANOTHER INTERESTING QUESTION
THAT WE'RE TRYING TO ADDRESS IS
WHAT ARE THE EFFECTS OF THESE
CRY YOWE PYRIN ASSOCIATED
PERIODIC SYNDROME MUTATIONS.
ALMOST ALL THESE MUTATIONS ARE
MAPPED TO THE NETWORKS -- NEK
DOMAIN BUT WE DON'T KNOW WHETHER
THIS AUTOMATIC CON FIR MAKAL
CHANGE IN THESE PROTEINS WOULD
ALREADY CAUSE DISPERSION OR YOU
STILL NEED A SECOND STIMULUS,
MAYBE A VERY WEAK STIMULUS IN
ORDER TO ACTIVATE THE PATHWAY
AND WE THOUGHT THIS COULD BE A
VERY INTERESTING ONE TO ADDRESS.
SO AS A SUMMARY FOR THIS PART OF
THE TALK, BASICALLY, WE SHOW
THAT HAD THE DIFFERENT NLRS. I
TALKED ABOUT THE NLRP INFLAM
SOME BUT THERE ARE OTHERS. WHAT
WAS STRIKING TO ME WAS THAT THE
DIFFERENT NLR PROTEINS ALL HAVE
THE SAME PROTEIN ORGANIZATION.
THEY ALL USE VERY DIFFERENT WAYS
TO ACTIVATE THE INFLAMMASOME.
FOR EXAMPLE I DIDN'T TALK ABOUT
NLRP 6 BUT IT GOES INTO A PHASE
SEPARATION. INTO DOUBLE
STRANDED. AND THIS IS THE
INITIAL SIGNALLING PLATFORM FOR
THE NLRP 6 INFLAM IF A SOME
ACTIVATION WHEREAS ONE USES A
CLEAVAGE IN ITS PROTEIN IN ORDER
TO RELEASE A TOXIC INFLAMMASOME
FORMING FRAGMENT TO INDUCE THE
ACTIVATION OF THIS PARTICULAR
NLR INFLAMMASOME WE LOOK FORWARD
TO LEARNING WHAT OTHER KIND OF
INFLAMMASOMES MAY DO. IN ITS
ABILITY TO INDUCE INFLAMMASOME
FORMATION. SO NOW I WILL TALK
ABOUT GSDMD IS A SUBSTRATE. WE
ACTUALLY KNOW NOW THAT GSDMD CAN
BE CLEAVED BUT OTHER ENZYMES I
THINK I PROBABLY HAVE A SLIDE IN
THE NEXT FEW SLIDES SO WE SHOWED
THAT GASDERMIN BINDS. WHERE ARE
THESE AT THE CELL? IN THE INNER
LEAFLET AND THAT'S WHY GASDERMIN
CAN PUNCH A HOLE FROM WITHIN AND
ONCE THEY GET RELEASED FROM THE
CELL THEY CANNOT ATTACK THE CELL
ANYMORE BECAUSE THERE'S NO
ACIDIC LIPIDS AND THE FACT THAT
IT CAN ALSO INTERACT WITH
CARDIOLIE PIN, THIS IS A LIPID
THAT'S PRESENT IN MITOCHONDRIA
AND ALSO SUGGESTS THAT
GASDERMINS CAN DAMAGE THE
MITOCHONDRIA AND THERE'S A LOT
OF EVIDENCE SUPPORTING THAT.
SO HERE'S A GASDERMIN FAMILY A
FAMILY THAT HAS BEEN THERE FOR
AWHILE. BUT DIDN'T DRAW A LOT
OF ATTENTION TO THE FIELD.
UNTIL THE DISCOVERY THAT THE
DOMAIN CAN PRODUCE THIS. AND IT
TURNS OUT ALL THE FAMILY MEMBERS
FROM A-F IF YOU WILL. THIS IS
AN F MEMBER. IT'S DOMAIN CAN
ALL INDUCE POOR FORMATION AND
PIE ROP TO SIS AND THEY CAN BE
ACTIVATED BY DIFFERENT KINDS OF
ENZYMES.
I LIST A FEW ON THE SIDE OF THIS
SLIDE. SO SO WHY PIE PROP SEW
SIS. IT IS VERY DIFFERENT FROM
APOP SEW SIS BECAUSE IT CAN
DANGER THE MITOCHONDRIA AND
THINGS CAN DO OUT AND IN. IT
CAN GO OUT. HMGB 1. A LOT OF
DIFFERENT DANGER MOLECULES CAN
GO OUT SO THEY'RE VERY IMMUNO
LOGICALLY HOT BY CONTRAST
APOPTOSIS IS IMMUNO LOGICALLY
COLD SO THEY DON'T REALLY ALERT
THE IMMUNE SYSTEM TO ATTRACT THE
ATTENTION THERE. SO OVER THE
YEARS WE HAVE SOLVED TWO
DIFFERENT GASDERMIN NT -- N
TERMINAL DOMAIN POOR STRUCTURES.
I JUST WANT TO DRAW YOUR
ATTENTION THAT GASDERMIN PORES
MAY BE FORMED IN TWO DIFFERENT
STEPS. ONE IS A PREPORE. WHAT
IS A PREPORE? THIS IS THE
YELLOW HERE IS A PREPORE
STRUCTURE. SO THE PREPORE IS
ALSO ALREADY O LIG MA RISED ON
THE MEMBRANE BUT IT HAS TO
INSERT INTO THE MEMBRANE SO AS
YOU CAN SEE HERE.
THE INSERTED PART OF THE
STRUCTURE IS A BETA BARREL.
THIS IS A LARGE BETA BARREL THAT
CONTAINS A LOT. 33 TIMES FOUR
BETA STRANDS THAT MAKES THIS
LARGE BETA BARREL SO THIS CAN BE
FORMED AND THEN INSERTION CAN
OCCUR IN ORDER TO FORM THE PORE
STRUCTURE. THIS WILL BECOME
IMPORTANT WHEN I TALK ABOUT THE
REMAINING PART OF THE TALK.
SOME OF IT UNPUBLISHED. SO THE
SUBUNIT OF GASDERMIN LOOKS
REALLY LIKE A LEFT HAND. THIS
IS THE INACTIVE STRUCTURE LIKE
THIS. THIS IS LIKE THE GLOBULAR
DOMAIN AND DURING INSERTION YOU
JUST HAVE YOUR FOUR FINGERS AS
THE TWO -- PAIRS OF BETA HAIR
PINS THAT GOES INTO THE
MEMBRANE. ONCE YOU INSERT YOUR
FINGERS LIKE THIS THE PORE IS
FORMED AND THE PORE BECOMES THIS
TRANSMEMBRANE PORE THAT CAN
RELEASE THE CYTOKINE OR OTHER
MOLECULES SO THIS IS A
SIMULATION OF THE EVENTS IN THIS
PREPORE TO PORE TRANSITION.
THIS -- YELLOW STARTS WITH A
PREPORE AND THEN THE STRANDS
INSERT INTO A PORE. SO ONCE WE
SOLVED THE PORE STRUCTURE THERE
WAS ONE QUESTION THAT BUGGED US
THAT IS THIS PARADOX. PROTO
LIEU KIN ONE WHICH IS ALSO VERY
SMALL CANNOT BE RELEASED FROM
LIVE CELLS SO THESE LIVE CELL
CONSIST STILL HAVE PORES ON THE
SURFACE AND THEY CAN RELEASE
MATURE ONE. WHICH IS 18 BUT
THEY CANNOT RELEASE THE PROTO
SAY TO SIGN AND THESE ARE THE
DATA SHOWN HERE SO THE PRODOES
NOT COME OUT BUT THE MATURE
CYTOKINES COME OUT SIZE-WISE
THEY ARE ALL ABOUT --
IN TERMS OF DIAMETER A QUARTER
OR LESS OF THE BIG PORE
STRUCTURE. LIKE THIS SIZE HERE
FOR A BIG PORE. BUT IT CANNOT
COME OUT. SO A PROMINENT
FEATURE OF THE PROCYTOKINE
THAT'S ABSENT IS THIS NEGATIVE
CHARGE TO SO THE PROPEPTIDE HERE
IS ACTUALLY EXTREMELY NEGATIVELY
CHARGED SO WE THOUGHT IS IT
POSSIBLE, THERE'S SOME KIND OF A
CHARGE SELECTION FOR THE
GASDERMIN PORE SELECTIVITY. SO
GASDERMIN D IF YOU LOOK AT THE
PI OF GASDERMIN IS ACTUALLY 8
POINT SOMETHING SO YOU WOULD --
AT LEAST I DID NOT, I NEVER
THOUGHT THIS COULD BE HAVE ANY
NEGATIVE CHARGE IN THIS PROTEIN.
IF YOU LOOK AT THE CHARGE
DISTRIBUTION ON THE ENTIRE PORE,
ALL THE STRONG POSITIVE CHARGES
ARE FACING THE MEMBRANE AND
THAT'S BECAUSE THEY'RE BINDING
ACIDIC LIPIDS AND INSIDE THE
PORE YOU SEE INSIDE THE PORE IS
ACTUALLY VERY ACIDIC. AND WHEN
YOU CALCULATE THE ELECTROSTATIC
POTENTIAL OF THE WILD TYPE
VERSUS MUTANT, IF YOU WANT
ACIDIC PATCH ONE OR ACIDIC PATCH
TWO AND THREE WHEN YOU CALCULATE
THE ELECTROSTATIC POTENTIAL YOU
DO SEE THAT WHEN YOU MUTATE.
YOU HAVE REDUCED AMOUNT OF
ACIDIC ELECTROSTATIC POTENTIAL
SO WE DID A LOT OF DIFFERENT
TESTS BUT I WANT TO SHOW YOU
THIS LAST TEST WE DID IN CELLS
IN WHICH WE USED THE CELLS AND
RECON.
>> THE STUDENT: CONSTITUTE WITH
EITHER THE WILD TYPE, SORRY, I'M
SO TERRIBLE WITH DRAWING THERE.
WILD TYPE OR THE ACIDIC PATCH
MUTANT OF GASDERMIN. AND THEN
STIMULATE THE CELLS WITH VERY
LOW DOSE NIGER RICIN FOR A LONG
TIME WHICH THE CELLS DO NOT DIE
AND LOOK AT THE RELEASE OF
INTERLIEU ONE ONE VERSUS THE
PROTEIN INTERLEUKIN ONE SO AS
YOU CAN SEE HERE WITH THE WILD
TYPE RECONSTITUTION YOU HAVE
ONLY THE MATURE CYTOKINE COMING
OUT VERY NICELY.
WHEREAS FOR THE CELLS THAT'S
RECONSTITUTED WITH THIS ACIDIC
PATCH MUTANT YOU SEE
PROINTERLEUKIN ONE COMING OUT
AND YOU ONLY HAVE A LITTLE BIT
OF THE MATURE INTERLEUKIN ONE
AND THAT'S BECAUSE THE
PROCYTOKINES ARE ALREADY OUT.
SO THEY DON'T HAVE A CHANCE TO
BE PROCESSED. REALLY SUGGESTING
THAT THIS SELECTIVITY FOR THE
RELEASE OF MATURE CYTOKINE AND
PERHAPS ALSO A LOT OF OTHER
THINGS IS GOVERNED BY THIS
CHARGE DISTRIBUTION WHAT WE CALL
ELECTROSTATIC FILTERING IN THE
GASDERMIN CARGO RELEASE. YEAH,
WHY DOES THE PORE HAVE THE
SELECTIVITY IS IT REALLY EVOLVED
ONLY FOR INTERLEUKIN ONE RELEASE
OR ALSO IMPORTANT FOR SOME OTHER
ASPECT OF THE BIOLOGY REMAINS
UNCLEAR. BUT ONE DISCUSSION
THAT I HAD I THINK WI. HE SAID
LOT OF DANGER MOLECULES ARE
POSITIVELY CHARGED WHICH MEANS
THEY CAN BE RELEASED FROM THE
PORE. SO WE SHALL SEE IF THAT
IS THE CASE WHEN WE PERFORM THE
EXPERIMENT WHICH IS ONGOING.
SO, I AM JUST LOOKING AT THE
TIME. OKAY, SO IN THE LAST
PART, I WANT TO TELL YOU AN
UNPUBLISHED STORY. ON THIS
QUESTION THAT HAS BUGGED US FOR
AWHILE. SO WE KNOW BACK IN 2016
WHEN WE DISCOVERED THAT
GASDERMIN CAN FORM PORES WE
MUTATED OTHER CYSTEINE RESIDUES
AND FOUND THAT C191A IS
DEFECTIVE. IF YOU TRANSFECT
THIS MUTANT YOU CANNOT KILL
CELLS BUT WE DIDN'T UNDERSTAND
WHY AND EVEN AFTER WE HAD THIS
STRUCTURE WE KNOW THIS RESIDUE
IS AT THE TIP OF THE BETA HAIR
PIN. RIGHT AT THE TIP BUT
THERE'S NO DULL SULFIDE AND THE
RESIDUE IS VERY FAR APART FROM
EACH OTHER IN THE PORE.
WE DON'T KNOW WHAT IS THE
FUNCTION OF THIS THING. SO
GIVEN THAT THIS IS A MEMBRANE
PROTEIN I ACTUALLY SUSPECTED
MAYBE IT IS LIP DATED BUT WE
REALLY DIDN'T DO AFTER IT UNTIL
A BRAVE POSTDOC DECIDES TO TRY
THAT EXPERIMENTALLY.
SO INDEED, TA TURNED OUT TO BE
THE CASE. THE CYSTEINE RESIDUE
IS POE MODULATED. THIS IS ONE
OF THE LIP DAGS FORM BUT POE
MODULATION IS I BELIEVE I
BELIEVE THE ONLY LIPID
MODIFICATION THAT IS REVERSIBLE
CAN BE REGULATED. SO WE USE A
NUMBER OF METHODS TO LOOK AT
THIS -- PAL MY TO MODIFICATION.
ONE IS CALLED ACYL BIOTIN
EXCHANGE WHICH BASICALLY YOU
MODIFY ALL THE FREE CYSTEINES IN
THE CELL. THEN YOU CLEAVE THIS
BOUND -- SO PAL MY TOLATION IS
WITH THE EXISTING RESIDUES USING
THIS HYDROXYL AMINE AND THEN
AFTER YOU TREAT THIS -- THESE
CELLS WITH HYDROXYL AMINE YOU
LEAVE THE THE GROUP WITH THE
FILE AND YOU CAN PUT IN ANYTHING
THAT YOU WANT A BIOTIN THAT HAS
A FREE FILE AND YOU CAN PULL IT
DOWN. SO AS YOU CAN SEE IT IS
MALL MY TOLATED BUT NOT C
TERMINUS. IT IS NOT PAL MY
TOLATED. OTHER GASDERMINS ARE
ALSO PAL MY TOLATED SO WE START
TO THINK THAT MAYBE THIS IS A
GENERAL PHENOMENA FOR THE ENTIRE
GASDERMIN FAMILY SO
INTERESTINGLY, IF YOU TAKE A
MACROPHAGE, YOU PRIME THE CELL
WITH LIE POE POLY SACK RAH, LPS,
YOU SEE A LITTLE BIT OF INCREASE
IN PAL MY TOLATION. WITHOUT IT
YOU SEE NOTHING, NOTHING CAN BE
DETECTED AND THEN WHEN YOU TREAT
A CELL WITH LPS PLUS NEM YOU SEE
A GREAT INCREASE. BOTH ON THE
CELLS AND THE N TERMINUS WHEREAS
AT LEAST UNDER THIS CONDITION
THERE'S NO GAME IN PROCESSING
SUGGESTING THAT THE ENTIRE
UPSTREAM EVENTS WERE NOT
AFFECTED. ONLY THE DOWNSTREAM
EVENTS ON THE LIPID MODIFICATION
OF GASDERMIN M.D. SO WE MAPPED
WHICH RESIDUE OF COURSE YOU KNOW
THE ANSWER ALREADY IS C191. I
COME BACK TO THAT BUT IT TURNED
OUT TO BE AN INTERESTING
RESIDUE. IT'S INVOLVED IN SO
MANY MYSTERIES ABOUT THIS
PROTEIN. SO WE THEN VALIDATE
THIS IN VITRO TO SEE WHETHER THE
PAL MY TOLATED GASDERMIN M.D. IS
MORE ACTIVATED. THIS IS A
LYSOSOME USING PURIFIED
PROTEINS. IF YOU PURIFY THESE
PROTEINS FROM BACTERIA, I
BELIEVE THE ORANGE HERE IS THE
WILD TYPE FROM BACTERIA. THE
GREEN IS THE C191 MUTANT FROM
BACTERIA. IN BACTERIA THAT
MEANS THEY'RE NOT PAL MY TO
LATED. YOU CAN SEE THIS IS THE
RITE OF LIPOSOME HAECAGE OF THE
RATE THAT THESE PROTEINS DPAJ
THE LIPOSOME TO RELEASE THE DYE.
BUT IF YOU USE MAMMAL YAN
GASDERMIN M.D. THIS IS ABOUT 50%
PAL MY TOLATED. ABOUT 50%. YOU
CAN ALREADY SEE ABOUT 10 FOLD
INCREASE IN THE RATE OF PAL MY
TOLATION SUGGESTING INDEED THIS
GROUP REALLY HELPED TO INSERT
GASDERMIN M.D. INTO THE MEMBRANE
SO FUNCTIONALLY, LIKE, THE SAME
THING, THE C191 A MUTANT IS
DEFECTIVE AND WHEN WE LOOKED AT
THEM BY CELLULAR IMAGING, WILD
TYPE, CAN GO TO THE CELL
SURFACE. YOU CAN SEE FROM THESE
STRUCTURES THAT'S ON THE
MEMBRANE, WHEREAS THE MUTANT,
THE C1 AND 191A MUTANT IS NOT
REALLY MEMBRANE LOCALIZED AT ALL
SO IT'S EFFECTING LOCALIZATION
AND PERHAPS ALSO PORE FORMATION.
SO THIS IS -- THIS I THINK IS
WHERE IT GETS REALLY
INTERESTING. SO BECAUSE THIS IS
ESTER BOUND SO WE WORKED WITH
OUR ESTER BOUND BEFORE AND WE
KNOW THAT ANY REDUCING AGENT,
ANYTHING THAT HAS A FREE FILE
CAN ATTACK THIS. AND DESTROY
THIS FILE ESTER. SO WE THOUGHT,
HUH, THIS PROBABLY MEANS THAT
THIS IS SENSITIVE TO THE REDOC
STATE OF A CELL. IF YOU HAVE
THIS IT WILL NOT STAY IF YOU
HAVE A MORE OXIDIZING
ENVIRONMENT MAYBE THIS WILL STAY
AND STAY LONGER SO THEN WE
THOUGHT, OKAY, MAYBE THEN THIS
WILL LINK IT TO THE GENERATION
OF ROS WITH CHANGES, THE REDOCK
STATE OF THE CELL SO WHAT WE DID
THEN WAS TO USE THE SAME TH
CELLS BUT ALSO INSTEAD OF USING
JUST LPS PLUS NIGER RICIN WE
ALSO TREATED CELLS WITH ROW TE
KNOWN IS ONE. ANTIMY SEEN A.
THESE TWO ARE INHIBITORS OF THE
MITOCHONDRIA RESPIRATORY
COMPLEX. ONE VERSUS THREE AND
THEY ALL INDUCE ROS IN THE
MITOCHONDRIA SO THESE TWO AS YOU
CAN SEE HERE INCREASES PAL MY
TOLATION BUT NO CHANGE IN THE
CLEAVAGE. IF YOU TREAT THE
CELLS IN CONTRAST USING ACETYL
CYSTEINE, THIS IS A REDUCING
AGENT YOU REDUCE THE AMOUNT OF
PAL MY TOLATION AMOUNT. AND
THIS IS ANOTHER INHIBITOR THAT
ALSO REDUCES THE CELLULAR ROS.
SO THIS IS ALREADY SAYING, OKAY,
IF YOU HAVE AN ROS ENHANCER YOU
HAVE MORE PAL MY TO LAGS. IF
YOU USE AN EXHIBIT TOR YOU HAVE
LESS. WHAT HAPPENED TO THE CELL
UNDER THOSE CAN BES, HAD THE ROK
REALLY INCREASED OR DECREASED.
THIS IS WHERE YOU TREAT THE
CELLS. THIS IS BY IMAGING THE
AMOUNT OF DWELLULAR ROS THAT YOU
OBSERVE OR THE MITOCHONDRIA OR
AS YOU SOEB. IF YOU TREAT A
CELL ALSO WITH THIS TWO YOU
REDUCE THE ROS. AND IF YOU
TREAT THE CELLS WITH THE ONE
THAT INCREASE PAL MY TOLATION
YOU ALSO GREATLY INKRECREASE TH
ROS SUGGESTING THEY'RE COUPLED.
ONE ENHANCING THE OTHER. SO
THEN WE LOOKED AT AMOUNT OF 23
HUMAN TRANSFERASES AND THEY'RE
ALL NAMED THIS GUY HERE BECAUSE
THEY HAVE A 2H AND THE CYSTEINE
AT THE ACTIVE SITE. WE TESTED
ALL OF THEM AND FOUND SEVERAL OF
THEM MAY ENHANCE THE PAL MY
TOLATION OF THIS. AND JUDGING
FROM THE -- SIT PROTEIN ATLAS?
WEST SIDE? WE CHOSE TO
INVESTIGATE FURTHER THESE FIVE
DIFFERENT PAL MY TO TILL
TRANSFERASES SO WHEN WE KNOCKED
DOWN ONE BY ONE THESE DIFFERENT
PAL MITO TRANSFERASES. YOU SEE
THIS IS IMPORTANT FOR THIS
TRANSFORMATION AND NINE IS
IMPORTANT AS WELL. SIMILARLY,
THE KNOCK DOWN OF THESE
TRANSFERASES ALSO REDUCE CELL
DEATH, MEMBRANE LOCALIZATION AND
EVERYTHING ELSE. SO THIS LED TO
OUR MODEL ABOUT WHAT ROS AND PAL
MY TOLATION MAY TOGETHER
REGULATE GASDERMIN D ACTIVATION.
SO SO THESE HAVE ALL BEEN SHOWN
TO LOCALIZE AROUND THE GOLGI AND
MAYBE ALSO THE ER AND MAYBE ALSO
OTHER LOCATIONS SO WE THINK THAT
WHEN GASDERMINS D IS NOT CLEAVED
MAYBE THE MAIN SOURCE OF PAL MY
TOLATION IS THROUGH THESE
CYTOSOLIC ONES AND WHEN YOU HAVE
HIGHER ROS YOU GET ADMINISTER.
SO THIS LITTLE SKIG L HERE IS
THE LIPID. AND THEN GASDERMIN D
CAN BE ACTIVATED OR CLEAVED BY
MATURE CAS CASE- 1 OR OTHER
CASPASES AND IF THE CLEAVED
GASDERMIN M.D. IS ALREADY PAL MY
TOLATED. IT WILL FORM A PREPORE
AND IMMEDIATELY TRANSIT INTO THE
PORE ITSELF. SO WHY DO I SAY
THAT? IF YOU LOOK AT THE
PREPORE AND WHERE THE
CALCULATION IS RIGHT AT THE TIP
OF THE INCERTIFICATED BITS THE
LIPID THAT IS -- THE LIPT THAT'S
ATTACHED TO THIS FINGERTIP HERE
I IMAGINE, AT LEAST, MAY HELP TO
DRAG THE INSERTION PROCESS SO
THAT EVERYTHING WILL GET INS
INSERTED INTO THE PORE. ANOTHER
REASON I SAY THIS IS JOHN'S
GROUP PUBLISHED A PAPER JUST
MAYBE ABOUT A YEAR AGO AND THEY
SHOWED THAT THIS MTOR RELATED
ROS IS IMPORTANT FOR THE
INSERTION OF THE PORE BUT NOT
FOR THE LOCALIZATION PER SE. SO
THIS I THINK, AT LEAST
MECHANISTICALLY CAN EXPLAIN
THAT. ROS WHICH EFFECTS SOME
MEMBRANE LOCALIZATION AND THE
FORMATION OF THE PREPORE BUT
MAYBE THE MOST IMPORTANT EFFECT
OF THIS IS TO DRAG THE PREPORE
INTO THE PORE TO FORM THIS
FUNCTIONING PORE. SO ONCE IT'S
CLEAVED IF THE PREPORE CAN ALSO
BE FORMED ON THE CELL MEMBRANE
THEN THE CELL MEMBRANE FORM OF
PAL MY TOLE TRANSFERASE,
ACTUALLY ONLY THE FIVE. OF ALL
THE TRANSFERASES FIVE IS THE
ONLY ONE KNOWN TO BE ON THE CELL
SURFACE AS WELL. I IMAGINE THE
FIVE WOULD BE THE MAJOR ONE THAT
WOULD CHANGE THE PREPORE TO A
PAL MODULATED PREPORE AND
FACILITATE ITS INSERTION. I'M
JUST LOOK AT MY TIME. SO IN
THEORY IT HAS BEEN SHOWN THAT
PAL MY TOLATED STUFF, THEY TEND
TO CLUSTER AND THIS IS BECAUSE
THESE LIPIDS, PALMITATE IS ALLEY
FATTIC. THEY ARE DOUBLE BOUND.
SO THEY'RE VERY ORDERED. THEY
CAN CAUSE PHASE SEPARATION IN
THE MEMBRANE SO THAT THE PORES
OR THE PAL MY TOLATED GROUPS
KNEW BECAMEMITOYLATED GROUPS KNW
BECAME PHASE SEPARATE INTO A
CLUSTERED ENTITY WITH MULTIPLE
PORES ALTOGETHER AND THIS IS
INDEED WHAT WE SAW ON THE LIGHT
IMAGING DATA. WE ACTUALLY DON'T
SEE A -- EVEN DISTRIBUTION OF
GASDERMIN ON THE CELL SUR
SURFACE. STRUCTURES.
WE WILL PROBABLY SEE A
CLUSTERING OF THE PORES. SO
INTERESTINGLY, A FEW YEARS AGO
WE PUBLISHED THIS DISULFUR RAM
WHICH IS A MODIFIER. GUESS WHAT
THEY ALL MODIFY. AND THE GROUP
AND KATE'S GROUP ALSO SHOWED
THESE OTHER MOLECULES AND THEY
ALL MODIFY CYSTEINE 191 AND THEY
ALL INHIBIT GASDERMIN M.D. AND
WE FIGURE THEY INHIBIT THE CELLS
BY COMPETING WITH PAL MY
TOLATION AND DIMETHYL FUME MA
RATE WORKS AND FUMARATE WORKS A
MOLECULE THAT GETS UPREGULATED
DURING REGULAR DAGS.
THIS MAY BE A NEGATIVE FEEDBACK
WITHIN THE CELL TO SUPPRESS
FURTHER INFLAMMASOME ACTIVATION
AFTER THE OCCURRENCE OF
INFLAMMASOME ACTIVATION. AS A
SUMMARY, I SHOWED YOU SOME DATA
NOW IN OUR P 3 PROJECT.
SUGGESTING THAT NLRP 3 IS
ACTIVATED THROUGH ONLY LARGE
CONFIRMATIONAL CHANGES THAT'S
AASSOCIATED WITH BINDING BUT
ALSO WITH TRAFFICKING AND
PERHAPS THESE STEPS CAN BE
TARGETED BY THERAPEUTICS.
AT THE MOMENT, ALL THE NLRP 3
DRUGS ARE TARGETING THE ADP
FORM.
TO KEEP IT IN THE ADP FORM SO IT
DOESN'T GET ACTIVATED AND THEN
WE SHOWED GASDERMIN PORE, EVEN
THOUGH IT'S SO LARGE WE WERE
PRETTY SURPRISED IT HAS SOME
SELECTIVITY. DESPITE ITS SIZE
IT DOES HAVE CERTAIN SELECTIVITY
BUT WE DON'T FULLY UNDERSTAND
WHY IT HAS THIS SELECTIVITY.
WHAT ELSE DOES IT MEDIATE INSIDE
A CELL? AND FINALLY, OUR
UNPUBLISHED DATA SHOWING THAT IT
IS ALSO MODIFIED BY THIS LIPID
GROUP AND I THINK THIS
CONNECTION TO ROS ALSO SAYS THAT
PYROPTOSIS IS NOT JUST ANY FORM
OF CELL DEATH. IT'S A CELL
DEATH OF THE LAST RESORT IF YOU
WILL. THE RESORTS ARE ALREADY
REALLY STRESSED OUT BY STRESS.
BY OXIDATIVE STRESS, FOR
EXAMPLE. AND THEN, YOU KNOW,
PYROPTOSIS IS ACHIEVED THROUGH
THIS CONDUIT SO MAYBE PYROPTOSIS
SHOULD REALLY BE AN OXIDATIVE
INDUCED CELL DEATH. WITH THAT,
LET ME THANK THE POST-SECONDARY
DOC STUDENTS AND I WANT TO
HIGHLIGHT MY COLLABORATION WITH
MY COLLEAGUE JULIE LIEBERMAN.
AND I THANK YOU FOR YOUR
ATTENTION.
>> (APPLAUSE).
>> HELLO, WOW. THANK YOU VERY
MUCH. LET'S HAVE ANOTHER ROUND
OF APPLAUSE.
>> THANK YOU.
>> THAT'S GREAT.
>> I DIDN'T GET ANY QUESTIONS ON
HERE. OH, WAIT, THERE'S ONE
HERE. SO LET'S --
>> OKAY.
>> JAY, SOME REPORTS HAVE
INDICATED THAT MITOCHONDRIAL
DATA RELEASE IS REQUIRED FOR
NLPR 3 ACTIVATION. DO YOU SEE
EVIDENCE FOR THIS?
>> YES, I TALKED TO MICHAEL
ABOUT THIS PRETTY EXTENSIVELY.
YEAH. WE TESTED PREVIOUSLY, TO
OUR INACTIVE TO SEE IF IT BINDS.
WE DID NOT SEE INTERACTION IN
OUR HANDS BUT NOW WE HAVE THE
ACTIVE SATE -- STATE TO SEE IF
IT BINDS AGAIN.
I WOULD LIKE TO MAKE A
CONNECTION BETWEEN THE
MITOCHONDRIA PATHWAY AND THE
TGN. I DON'T KNOW HOW TO
RATIONALIZE THIS WHOLE THING YET
BUT HOPEFULLY WE'LL GET THERE.
>> GREAT. OTHER QUESTIONS?
>>THIS IS PETER. YOU DESCRIBE LUCID
EXTENSIVELY IN THE VESICLE
TRAFFICKING AND TGN. BUT THE
PRIMARY FUNCTION IS ACTIVATE PER
CASPASE, RIGHT? SO DO YOU SEE
ANY EVIDENCE? HOW PRO-CASPASE-1
GETS INTERACT WITH THE
INFLAMMASOME AND THEN
SUBSEQUENTLY --
>> YEAH. YEAH. YEAH. SO THAT
ACTUALLY I THINK THAT
QUESTION -- WELL, IT MAY BE MORE
COMPLEX BUT IN OUR I THINK 2016
PAPER OR SOMETHING SO WE LOOKED
AT HOW IT INTERACT. SO IT HAS A
DOMAIN ON THE TERMINUS AND
INTERACT WITH THE C DOMAIN AND
ONCE THIS INTERACTION OCCURS
THIS WILL BE BROUGHT TOGETHER
AND THEN THEY DIE AMOR RISE SO
THEN THEY CAN THEN
AUTOPROCESS -- TRANSAUTOPROCESS
TO BECOME CLEAVED AND ACTIVE.
SO MANY MY HEAD THIS IS WHAT'S
HAPPENING. BUT I THINK THE
STORY PROBABLY IS MUCH MORE
COMPLEX. WE HAVE SOME
UNPUBLISHED DATA WHICH IS STILL
NOT COMPLETELY CLEAR BUT WE
THINK THAT THE DIRECT
RECRUITMENT OF PROCYTOKINES, 18
AND L 1 IS NOT THAT SIMPLE.
IT'S ACTUALLY, YOU KNOW, IT'S --
MORE COMPLEX THAN JUST A SIMPLE
RECRUITMENT. WE THINK THERE'S
MORE STEPS THAT'S INVOLVED IN
THERE BUT WE DON'T KNOW ABOUT
THE SUBSTRATE RECOGNITION YET
BUT I THINK WE'RE PRETTY CLEAR
ABOUT THE CASPASE ACTIVATION BUT
WE DON'T KNOW ABOUT SUBSTRATE
RECRUITMENT. AND AS YOU KNOW,
FUNCTIONAL GROUP AND ALSO I
THINK THE GROUP HAVE SHOWN
THAT -- HAS SHOWN THAT GASDERMIN
D NEEDS THIS SITE TO BE
RECRUITED TO CASPASE-1. SO IT'S
NOT JUST AN ACTIVE SITE
INTERACTION BUT ALSO AN ACYL
SITE INTERACTION. THIS IS MORE
IMPORTANT THAN THE MOTIF
RECOGNITION. SO I -- I THINK
OUR DATA MAY BEGIN TO SHOW THAT
THE -- SOME KIND OF AN XO SITE
INTERACTION IS ALSO FOR
PROCYTOKINE PROCESSING.
>> MY UNDERSTANDING IS THAT
ACTIVATION, CASPASE DOES REQUIRE
ASSEMBLY OF INFLAM SOME SO IT
POSSIBLE TO PUT A CATALYTICALLY
DEAD CASPASE CONSTRUCT INTO
YOUR -- ANY.
>> YES, ABSOLUTELY, YEAH. M-HM.
WE THINK THE DIE AMORIZATION
WILL STILL OCCUR WITHIN THE
FILAMENT, YEAH.
>> YES.
>> THANK YOU SO MUCH FOR
WONDERFUL TALK, DR. WU. I AM
ACTUALLY FASCINATED ABOUT THE
PAL MY TOLATION AND YOUR DATA
BEAUTIFULLY EXPLAINS WHAT
HAPPENS WHEN IT IS ACTIVATED. I
WOULD LIKE TO ASK IN CASE OTHER
INFLAMMASOMES ARE ACTIVATED. DO
YOU THINK THE SAME THING OCCURS
TO GASDERMIN M.D. BECAUSE --
>> WE HAVEN'T TESTED BUT I THINK
SO. I THINK BECAUSE THIS IS A
PHYSICAL PRINCIPLE. SO I DON'T
THINK IT'LL BE DIFFERENT.
UNLESS THERE'S OTHER MECHANISMS
TO COMPENSATE FOR THAT. I
IMAGINE OUR INFLAMMASOME
ACTIVATION RAISE ROS. I DON'T
KNOW HOW AND THAT WILL CAUSE
GASDERMIN M.D. AT LEAST THAT'S
WHAT I IMAGINE.
BUT WE HAVEN'T TESTED THAT.
>> OKAY. ANOTHER QUESTION,
IS -- DO YOU THINK THAT IN OTHER
GASDERMINS, IS THE CYSTEINE
RESIDUE CONSERVED.
>> NO, IT IS NOT CONSERVED BUT
OTHER GASDERMINS THAT WE'RE ABLE
TO TEST SO FAR ARE
PALMITOYLATED. THERE'S ONLY ONE
WE COULDN'T TEST BECAUSE IT
DIDN'T EXPRESS WHICH IS
GASDERMIN C BUT EVERYTHING ELSE
WE TESTED OR PALMITOYLATED. AS
A STRUCTURALIST I LIKE SIMPLE
THINGS. SO I THINK THEY --
THERE'S GOT TO BE SOME
COMMONALITY AMONG ALL THE
DIFFERENT GASDERMINS.
>> OKAY. THANK YOU.
>> YES.
>> VERY NICE TALK WITH THE NLRP
3 SUMMARY SLIDE YOU SHOW THAT
HAD THE INHIBITED BUT CAGED FORM
IS WHAT GETS TRAFFICKED. HOW
DOES THAT HAPPEN? WHY DOES IT
HAVE TO BE IN THAT CAGED
INHIBITED FORM? HOW IS THE
SELECTIVITY ENFORCED?
>> WE ARE TRYING TO FIGURE THAT
OUT. WE DON'T KNOW. SO I THINK
THE CAGE IS REQUIRED FOR THE
GOLGI BINDING BECAUSE YOU HAVE
MULTIVA LENIENCY. IF YOU JUST
HAVE ONE, YOU KNOW, POSITIVELY
CHARGED PATCH IT'S PROBABLY NOT
ENOUGH YOU NEED THE WHOLE THING.
SO BUT WHY DOES THE CAGE INDUCE
GOLGI DISPERSION? THAT WE DON'T
KNOW. AND THAT'S A QUESTION WE
REALLY WANT TO ADDRESS, YEAH.
>> OKAY. I'M POSTDOC IN MAX
LAB. SO I'M WONDERING IF
GASDERMIN D IS ALMOST SELECTIVE
FOR MEMORY.
>> I DIDN'T MENTION THAT BUT
CERTAINLY MITOCHONDRIA.
>> TWO LAYER MEMORY.
>> YEAH. SO THERE'S -- MAYBE
THOSE ARE NOT PUBLISHED BUT I
THINK MITOCHONDRIA DAMAGE BY
GASDERMIN HAS BEEN IMPLICATED.
MAYBE NOT DIRECTLY SHOWN BUT
IMPLICATED. YEAH. SO THE IDEA
IS THAT THE LIKING THAT'S IN THE
MEMBRANE OF THE MITOCHONDRIA CAN
BE EXTERNALIZED TO THE OUTSIDE.
IN FACT, THE SCRAMBLING THAT'S
IMPORTANT FOR THIS PROCESS IS
IMPORTANT FOR GASDERMIN D
LOCALIZATION TO THE
MITOCHONDRIA.
>> ANOTHER QUESTION IS AS YOU
SAID, THE GASDERMIN D BE CLEAVED
INTO AN N TERMINAL. WHAT IS THE
FUNCTION.
>> I HAVE NO IDEA WHAT THE C
TERMINAL DOES. I HAVE TO IDEA.
YEAH. YEAH.
>> DO THEY FORM, LIKE, A DIMER
OR ONLY THE -- THE C TERMINAL
RELEASE AND THE FUNCTION --
>> YEAH, WE DON'T -- SO THE ONLY
FUNCTION I KNOW ABOUT FOR THE C
TERMINUS IS TO SUPPRESS THE N
TERMINUS FOR SURE IN THE PROTEIN
IT DOES THAT. ONCE IT'S
RELEASED WHAT DOES IT DO? I
HAVE NO CLUE. I HAVE NO IDEA
WHAT IT DOES AFTERWARDS: YEAH,
MAYBE IT'S NOT FULLY -- OH, SO I
WANT TO MENTION ONE THING. SO
GASDERMIN D I DIDN'T TALK ABOUT
THAT PART OF OUR WORK BUT
GASDERMIN D IS EXPRESSED WIDELY
IN CELLS. NOT JUST IN
MACROPHAGES SO THAT WAS REALLY
STRANGE. IF IT'S DOWNSTREAM OF
INFLAMMASOME WHY IS IT EXPRESSED
IN OTHER CELLS? FOR EXAMPLE, IN
A LOT OF CANCER CELLS IT'S
EXPRESSED. SO I DON'T KNOW, BUT
I IMAGINE GASDERMIN D HAS OTHER
FUNCTIONS. MAYBE THAT WILL HAVE
TO DO WITH THE C TERMINAL
DOMAIN. IT'S ANYONE'S GUESS I
THINK AT THIS POINT. YEAH.
>> SO THE CLEAVE, THE GASDERMIN
D IS ONLY ONE --
>> NO, THERE'S ALSO OTHER ONES.
>> LIKE RUN DOWN SOME.
>> DEPENDS ON WHICH ONE. B I
THINK. NO, THAT'S CLEAVING --
THAT'S ACTUALLY CLEAVING E.
IT'S BETTER I GO THROUGH MY
TALK. I ACTUALLY DON'T REMEMBER
THE C. OH -- I DON'T KNOW HOW
TO GO BACK, OKAY. SO IT'S HERE.
CASPASE A ALSO CLEAVES D. AND
THE KNEW TRUE FILL ELASTASE
CLOOEAGE OF D IS ACTUALLY
IMPORTANT FOR ME TO SIS. THIS
NEUTROPHIL EXCLUSION OF NUCLEAR
DNA AND CASPASE A THROUGH THE
APOPTOTIC PATHWAY CAN ALSO
CLEAVE GASDERMIN D.
>> THANK YOU.
>> YEAH.
>> (SPEAKER FAR FROM MIC).
>> YEAH. IT'S ABOUT -- I
MEASURED THIS BEFORE AND NOW I
DON'T KNOW IF I REMEMBER WHEN I
WAS WRITING THE FIRST PAPER I
MEASURED IT. IT'S ABOUT A
SLIGHTLY COMPRESSED DOUBLE LA
LAYER. IT'S LIKE SLIGHTLY
COMPRESSED I WOULD SAY. SO IT'S
POSSIBLE IT CAUSES SOME MEMBRANE
CURVATURE AS WELL.
>> (SPEAKER FAR FROM MIC).
>> IT IS GOING THROUGH THE
MEMBRANE ALREADY. IF YOU CHANGE
THE HYDROPHOBIC BITS. YEAH.
I'VE NOT DONE THAT. YOU WONDER
WHETHER THAT WILL CHANGE THE
SELECTIVITY. YEAH. IT MAY.
YEAH.
>> ONE MORE QUESTION?
>> ONE MORE FINAL QUESTION.
>> THANK YOU FOR THE TALK. I
HAVE ONE QUESTION. WHY THE
SAMPLE TEST? I WANT TO KNOW
THE --
>> YEAH. YEAH.
>> THE MOLECULAR BASIS OF THE
FUNCTION.
>> YEAH. YEAH. SO WE FOUND THE
REASON THAT WE INVESTIGATED WAS
BECAUSE WE SEE THESE SINGLE
SPECS IN A CELL. WE WERE, LIKE,
WHY? WHY IS IT ONLY ONE? WHY
IS EVERYTHING COALESCE TO ONE?
AND IT'S PARA NUCLEAR SO WE WERE
JUST, LIKE, IT LOOKS LIKE MTOC
SO WE TESTED BY
IMMUNOFLUORESCENCE AND INDEED
THEY ARE ONLY MTOC AND THEN WE
TESTED MICROTUBULE SENSITIVITY,
ARE THESE THINGS SENSITIVE TO
MICROTUBULE STRUCTURES AND THIS
IS ALSO BEFORE US. EVEN JURG
TESTED THESE AND THEY ALL
DISRUPT THE ACTIVATION SO WE
RETESTED THOSE AND ALSO MAPPED
THE INHIBITOR ALSO INHIBITED IN
RP 3 AND THE ADAPTER ADAPTER
WHICH IS HDAK 6 WHICH BINDS ALSO
INHIBIT THE MICROTUBULE
TRANSPORT. WHAT'S THE REASON
FOR THAT? GOOD QUESTION. MY
RATIONALE FOR THAT IS THIS
PATHWAY IS ALSO SIMILAR TO THE
SO-CALLI
SO-CALLING A ING A RAH SOME
PATHWAY.
THEY ARE TOO BIG I THINK TO BE
DEGRADED BY THE PROTEOSOME SO
THEY GET TRANSPORTED TO THE MTOC
AND AT THE MTOC THERE'S USUALLY
A LOT OF DONOR MEMBRANES. YOU
HAVE MITOCHONDRIA RIGHT THERE.
THERE'S LOTS OF THINGS THERE.
SO YOU FORM OTHERS. SO THAT'S
HOW YOU DEGRADE THESE PROTEINS
SO IN OUR CASE, WE ALSO SAW THE
SPEC. THIS WAS THE OLD WORK IN
OUR SPEC THEY'RE ENCLOSED
OFTENTIMES BY DOUBLE MEMBRANES.
SO WE THINK IT GOES THERE TO
REGULATE AND IF WE TREAT WITH
3MA THAT INHIBIT THE PUMP THAT
GOES FROM HERE TO HERE ARE HUGE.
THEY HAVE THIS GIGANTIC AND IT'S
AT THE LEVEL OF INFLAMMASOME
ACTIVATION OF THE CELL. SO WE
THINK WE GO THERE TO ACTIVATE
AND GO THERE TO BE REGULATED.
>> YES. THANK YOU. I THINK YOU
ARE -- THERE SHOULD BE A
MECHANISM RECRUITING THE RP 3 OR
THE MECHANISM TO THE MTOC. I
THINK THERE SHOULD BE A
MECHANISM THERE.
>> WE THINK IT'S ON THE VESICLE.
THAT'S WHAT WE THINK. THE
DISPERSION. WE THINK IT'S THE
VESICLES THAT GETS TRANSPORTED
BUT WE'RE NOT 100% SURE. THE
REASON WE SAID IT'S THE VESICLE
THAT GETS TRANSPORTED IS BECAUSE
TGM MARKERS BECOME ON THE MTOC
WHEN YOU HAVE INFLAM SOMES SO WE
FIGURED THAT THAT MUST BE THE
VESICLE THAT GETS TRANSPORTED.
>> THANK YOU VERY MUCH. THAT
WAS REALLY FANTASTIC.
>> THANK YOU.