GOOD AFTERNOON, EVERYONE,
WELCOME TO THE WEDNESDAY
AFTERNOON LECTURE SERIES I AM
IAN FRAZIER FROM NIAID AND IT'S
A GREAT PRIVILEGE FOR ME TODAY
TO INTRODUCE OUR SPEAKER
DR. JAMES CHEN WHO'S DONE
REMARKABLE WORK IN HIS CAREER IN
REBALDING PATHWAYS AND PATHWAYS
THAT TRIGGER RESPONSES, AND FOR
THOSE THAT WATCH THE VIDEOCAST
WE ENCOURAGE TO YOU PARTICIPATE
IN THE DNA AFTER THE SESSION,
YOU CAN DO SO BY USING LIVE
FEEDBACK LINK BELOW THE
VIDEOCAST WINDOW AND WE'RE
OFFERING CONTINUING
METRICSICATION--[LAUGHTER]
CONTINUING MEDICAL EDUCATION,
50095.
SO BACK TO OUR SPEAKER AND THIS
AFTERNOON, SO JAMES CHEN IS THE
GEORGE Mc GREGOR DISTINGUISHED
CHAIR IN BIOMEDICAL SCIENCE AND
THE DIRECTOR OF INFLAMMATION
RESEARCH CENTER AT THE
UNIVERSITY OF TEXAS SOUTHWESTERN
MEDICAL CENTER.
HE RECEIVED HIS Ph.D.
PRACTICES THE STATE UNIVERSITY
OF NEW YORK AND AFTER WORKING IN
THE SALT INDUSTRY, HE STARTED
ACADEMICS AT 1997 AND ROSE TO
FULL PROFESSOR THERE BY 2 THIS
HAPPENED 5 AND BECAME A HOWARD
HUGHES INVESTIGATOR IN THAT SAME
YEAR AND EARLY IN HIS CAREER,
JAMES REALLY MADE SOME MAJOR
DORPHYS IN HOW CELLS USE
DIFFERENTLY LINKED CHANGE UP OF
SMALL PROTEIN UBIQUITIN AND
REGULATE CELLULAR RESPONSES
SHOWING IN PARTICULAR CASE 63
LINK CHAINS OF UBIQUITIN WOULD
MARK PROTEINS AND NOT FOR THE
TYPICAL DEGRADATION PATHWAY BUT
INSTEAD TO LEAD TO KIND OF
SPECIFIC SIGNATURES MALAND
OUTCOMES.
HE THEN WENT ON TO DISCOVER AND
DESCRIBE THE MITOCHONDRIA
PROTEIN MAVS WHICH IS A CRITICAL
COMPONENT IN THE HOST CELL
RESPONSE WITH THE VIRUSES SUCH
AS EBOLA AND CONTINUING THE
THEME OF CELLS TO NUCLEIC ACIDS,
JAMES IS SAID TO HAVE TACKLED
THE MYSTERY OF THE PROTEIN FOR
MICROBIAL DNA AND THED LESTO A
MAJOR DORPHY PRACTICES JAIM'S
LAB OF THE CYCLIC GMP, AMP
SYNTHASE OR C-GAS AND A PAIR OF
REALLY BEAUTIFUL BACK-TO-BACK
PAPERS IN THE ISSUE OF SCIENCE,
HIS LAB SHOWED A NOVEL SECOND
MESS ESMGER MOLECULE CALLED
CGAMWHICH TURNED OUT TO BE THE
NUCLEOTIDIFYINGER TO MICROBIAL
DNA, PATHWAY REALLY GOES WELL
YAWBD MICROBIAL INFECTION, AS
DNA SENSOR FUNCTION IS CRITICAL
IN 19 DISEASES INCLUDING CANCER,
AND AUTOIMMUNITY.
SO IN RECOGNITION OF THIS REALLY
GROUND BREAKING WORK IN 2019
JAMES RECEIVED THE BREAK THROUGH
PRIZE IN LIFE SCIENCES AND IN
AKIGZ TO THIS HIGHLY PRESTIGIOUS
AWARD HE RECOGNIZED WITH THE
BILL PAUL DISTINGUISHED AWARD
AND AUTOIMMUNITY, THE PRIZE IN
BIOMEDICAL CONSCIENCES FROM THE
FNIH AND THE [INDISCERNIBLE]
ROSE HOROWITZ PRIZE THIS YEAR
FROM COLUMBIA, LAST BUT NOT
LEAST, JAIM SYSTEM A MEMBER OF
THE NATIONAL ACADEMY OF SCIENCES
AND TODAY IS THE DARK SIDE OF
DEFICIENCY NA HAVE THEM IN
SYSTEMS SINCE HIS DINITIATIVESA
AS A DANGER SIGNAL SO PLOAZ JOIN
ME IN WELCOMING YAIMS TO THE
NIH.
[ APPLAUSE ]
OKAY, THANK YOU VERY MUCH IAN
FOR A VERY KIND INTRODUCTION.
I KNOW IT IS REALLY A GREAT
HONOR FOR ME TO COME HERE AND TO
GIVE THIS TALK.
SO I WANT TO THANK ALL OF YOU
FOR ATTENDING THIS TALK, EITHER
IN PERSON OR VIRTUALLY.
I ALSO WANT TO THANK THE
CONGRESS FOR KEEPING THE
GOVERNMENT OPEN AND THAT GIVES
ME A SMALL WINDOW OF OPPORTUNITY
TO COME HERE AND TELL YOU ABOUT
OUR WORK.
YOU KNOW JUST 3 WEEKS EARLIER OR
3 WEEKS LATER THIS WOULD BE
QUITE [INDISCERNIBLE].
SO TODAY I WILL TELL YOU ABOUT A
DARK SIDE OF DNA.
MOST PEOPLE HERE WORK ON THE
BRIGHT SIDE OF DMA AND THAT IS
HOW DNA FUNCTIONS AS A GENETIC
MATERIAL, HOW DNA IS REPLICATED
AND TRANSCRIBED BUT DNA ALSO HAS
A DARK SIDE AND THAT IS IT CAN
TRIGGER IMMUNE RESPONSES AND
SOMETIMES IT CAN CAUSE
AUTOIMMUNE DISEASES AND IT HOLDS
UP OTHER DISEASES.
ALL RIGHT SO THESE ARE THE MAJOR
PLAYERS IN MY TALK, THESE ARE
THE SWROAT ENZYMES C-GAS WHICH
IS ACTIVATED BY BYPASSING TO THE
DOUBLE STRANDED DNA AND THE
ACTIVATED ENZYME CONVERTS
CTP AND ATP TO THE CYCLIC CAMP
FOR SHOWS AND IT BYPASSEDS IN
ACTIVATES ER-MEMBRANE PROTEIN
CALLED STAIN, ASK STAIN
SIGNALING A CASCADE LEADING TO
INNATE IMMUNE RESPONSE.
SO THIS IS THE TIELT OF MY TALK.
FIRST I WILL BRIEFLY TALK ABOUT
HOW BEE DISCOVER CGAS IN CGAMP
AND I HOPE TO USE THESE AN
EVIDENCE TO ILTRAIT TO STUDENTS
AND POST DOCS NAIN THIS SINGLE
CELL GENOMIC ERA IT'S POSSIBLE
TO MAKE AN IMPORTANT DISCOVERY
USING OLD FASHIONED
BIOCHEMISTRY.
OKAY?
AND THEN WE WILL TALK ABOUT 2
RECENT STORIES, FIRST 1 CGAS
STAIN SIGNALING IN ANIMAL CELLS
AND THEN I WILL TALK ABOUT
BACTERIAL CGAS IN ANTIIMMUNE
DEFENSE.
SO WHY DO WE WANT TO START AN
IMMUNE SENSING OF DNA?
AND WITHIN THAT RECOGNITION OF
FOREIGN DNA IS 1 OF THE MOST
FUNDAMENT AMILLIO MECHANISMS OF
HOST DIFFERENCE AND EVEN
BACTERIA CAN DO THAT, THE
CLASSICAL EXAMPLES ENCLUED
RESTRICTION ENZYMES THAT CUT THE
CURE OF DNA OR THE PLASMA OF DNA
AND A MORE RECENT EXAMPLE IS THE
CRSPR SYSTEM WHICH IS THE
ADAPTER IMMUNE SYSTEM THAT CUTS
FOREIGN DNA.
IN HUMANS DNA WAS KNOWN TO
STIMULATE IMMUNE RESPONSES MORE
THAN A HUNDRED YEARS AGO.
--THERE ARE SURGEONS IN FRANCE
AND GERMANY WHO INTRODUCE THE
ABDOMINAL CAVITY OR UNDER THE
SKIN OF THE PATIENTS EITHER 1
[INDISCERNIBLE] DNA, AND THE
OBLIGATIONS YECT WITH THE
PROTECTIVE AMIOFAIG O SIGHTS TO
WARD IT OFF EMPLOY SO DNA WAS
KNOWN TO TIMMULATE IMMUNE
RESPONSES 40 YEARS BEFORE IT WAS
KNOWN AS GENETIC MATERIAL BUT WE
STILL DON'T QUITE UNDERSTAND HOW
DNA STIMULATE IMMUNE RESPONSES
UNTIL QUITE RECENTLY.
SO WHY DO WE WANT AN IMMUNE
SYSTEM THAT RECOGNIZE D NA?
FIRST OF ALL THIS WOULD BE
EXCEPTION ALGORITHMS, FIRST BY
THE VIRUSES RECOGNIZED BY THE
[INDISCERNIBLE] PATHWAY THAT I
WILL TALK ABOUT IN JUST A
MINUTE, ALL MICROORGANISMS
INCLUDING VIRUSES, FUNGI AND
PARASITES THEY ALL HAVE DECKER
NA AND OR REQUIRE DNA IN THEIR
LIFE CYCLE AND THAT PROVIES AN
ALMOST UNIVERSAL MECHANISM TO
DETECT MICROBIAL INFECTIONS.
AND SECONDLY, RECOGNITION OF DMA
OR RNA, IN THIS MICROORGANISMS
PROVIDE A MECHANISM TO DETECT
THOSE MICROORGANISMS THAT LIVE
AND REPLICATE INSIDE OUR CELLS
EMPLOY WE ALL KNOW PART OF THE
RECEPTORS ARE IMPORTANT IN
INNATE IMMUNITY BUT THESE ARE
PROTEINS WITH A LIGAND BINDING
INSIDE THE SOILS AND SOME ARE IN
THE ENDOSTUDIES OF MULTIPLE
ENDOCRINAL MEMBRANE WITH THE
LUMEN OF THEEND SOSOME WHICH IS
EQUIVALENT TO OUTSIDER CELLS.
SO TOLL RECEPTORS RECOGNIZE THIS
OUTSIDE OF THE CELLS BUT THEY
ARE BLIND TO THIS BOX.
THEY HAVE SUCCESSFULLY INVADED
OUR CELLS AND REPLICATED INSIDE
OUR CELLS.
AND THE RECOGNITION OF THIS DNA
OR ROBBER NA FROM THIS BOX, THEN
PROVIDES A POWERFUL MECHANISM TO
DETECT THESE REPLICATING
MICROORGANISMS.
BUT THIS SYSTEM IS LIABILITY
BECAUSE OUR CELLS HAVE LOTS OF
DNA AND OUR SOLUTION TO THIS
PROBLEM IS TO KEEP OUR DNETWORKA
OUT OF THE CYTOPLASM IN
EUKARYOTIC CELLS.
WE KEEP OUR DNA IN THE
NUCLEOTIDES EUS AND IN THE
MITOCHONDRIA AND IN THE NUCLEUS
WE WRAP OUR DNA AROUND HIPPO
STUDIES OF MULTIPLE ENDOCRINES
TO CREATE 94 ORGANISMS.
BUT THIS PERSON IS NOT PERFECT
AND WHEN IT BREAKS DOWN IT CAN
LEAD TO A WIDE VARIETY OF CAUSES
AND IT'S ALSO IMPORTANT IN
CANCER.
SO BEFORE I TALK ABOUT A DNA
SENSING PATHWAY, I WILL BRIEFLY
TALK ABOUT THE RNA MESSAGING
PATHWAY WHICH MY GROUP HAS ALSO
BEEN WORKING ON.
SO WHEN AN ANTIVIRUS, OR
SARS-COV-2, INFECT OURSELVES,
THE VIRAL RNA IS A DETECTED BY
THESE HELIXES CALLED
[INDISCERNIBLE], AND THEN THESE
SENSOR PROTEINS INTACT WITH THIS
MITOCHONDRIA AUTOMEMBRANE
PROTEIN THAT WE IDENTIFIED MANY
YEARS WHICH WE CALL MAVs, IT
STANDS FOR MITOCHONDRIA
ANTIVIRAL SIGNALING, WE ALSO
CALL IS MAVs, BECAUSE WE'RE IN
DALLAS, AND WHE WE DISCOVER THIS
MOLECULE IT HAPPENED THAT DALLAS
MAVERICKS WAS PLAYING IN THE NBA
FINAL SO THAT'S HOW WE GOT OUR
NAME EMPLOY --NAME.
SO THIS MITOCHONDRIA SITS ON THE
MEMBRANE AND IT ACTIVATES THESE
KINASES IN THE CYTOSOL, PKP1
WHICH ACTIVATES IL3 AND THESE
TRANSCRIPTION FACTORS MOVE INTO
THE NUCLEUS INTO THE OTHER
CYTOKINES AND THIS IS HOW WE
FIGHT RNA VIRUS INFECTION.
AND AS I MENTIONED EARLIER DNA
FROM BACTERIA OR VIRUSES, THEY
CAN ALSO LEAD TO TYPE 1
INTERFERON PRODUCTION.
BUT A SENSOR OF DNA WAS NOT
KNOWN FOR A LONG TIME AND DOWN
STREAM IN THE SENSOR, THE
REMEMBER PROTEIN CALLED STAIN
WHICH WAS DISCOVERED BY
[INDISCERNIBLE] BARBER IN MIAMI
[INDISCERNIBLE] IN CHINA.
AND STAIN THEN ALSO ACTIVATE THE
IKCASHING AND [INDISCERNIBLE] IN
THE FIELDS.
SO WHAT WAS THE SENSOR IN THERE
WAS A LOT OF INTEREST IN FINDING
THIS DNETWORKA SENSOR ABOUT A
DECADE AGO EMPLOY SO WE TOOK A
BIOCHEMICAL APPROACH TO TRY TO
IDENTIFY THIS DNA SENSOR AND IN
BIOCHEMISTRY, THE MOST IMPORTANT
FIRST STEP IS TO SET UP A CELL
FREE SYSTEM THAT RECAPITULATES
WHAT'S HAPPENING INSIDE CELLS.
SO THIS IS HOW WE DID KNOW ARK
SAY, WE EXTRACTED IT INTO THE
CELL PATHWAY, AND WE USE SHRNA
TO KNOCK DOWN TAIN SUCH THAT WE
CAN TRAP EFFECTOR THAT IS
ACTIVATED BY DNA FUNCTION
UPSTREAM OF STING, SO SUPPOSEDLY
PURE DNA SENSOR WOULD BE
ACTIVATED BY DNA AND IF WE WELL,
YSE THE CELLS, PREPARE THE CELL
LIESATE, THE CELL WILL CONTINUE
TO THE FACTOR THAT IS ACTIVATED
BY DNA.
WE THEN DID A CELL ARK SAY INTO
ANOTHER CELL, IN THIS CASE,
[INDISCERNIBLE] CELLS AND THEY
HAVE A STING PATHWAY AND WE USE
THE TOXIN CALLED [INDISCERNIBLE]
TO PUT IT ON THE MEMORY RESPONSE
BREAN SO WE CAN DELIVER THESE
FACTORS INTO THE CELLS.
STING IS USING ER PROTEINS SO IT
CAN BE THE GET OUT OF THE CELLS,
AND HAD IT'S ACTIVATED IT WILL
SIGNAL A CASICATE IN
DEPHOSPHORYLATION OF
DIMERIZATION OF IREFRESH YOUR
RECOLLECTION REFRESH YOUR
RECOLLECTION F3, SO WE CAN RUN
THIS TO DETECT THE IRF3 DIMER.
SO THIS ASSAY, EVENTUALLY
WORKED.
IT TOOK ABOUT 2 YEARS TO GET IT
TO WORK AND IT WAS WORKED OUT BY
A VERY TALENTED BIOCHEMIST IN
THE LOB JOSH SUN.
SO, SO FINALLY THIS ASSAY
WORKED.
HERE IF WE USE EXTRACTS FROM DNA
TRANSVECTED CELLS AND DELIVERED
EXTRACT INTOS POLYMERIZED CELLS,
WE CAN SEE THESE IL3 DIMER AND
THESE ARE NEGATIVE CONTROLS.
SO NOW, WE HAVE ASSAY, CELL-FREE
ARK SAY, AND THE PAN WAS TO TART
TO FRACTIONATE A CELL EXTRACTS
TO PURIFY THE ACTIVATOR BUT
BEFORE WE DID A FRACTIONATION,
WE ASK A SIMPLE QUESTION, IS
THIS ACTIVATOR A PROTEIN OR NOT.
SO WE TOOK THE EXTRACTS AND HEAT
IT UP TO 95-DEGREE, WITH THE
PROTEINS, SPUN IT DOWN WITH THE
PROTEINS AND DELIVER THE
SUPERINATENT INTO THE CELLS.
AND TO OUR SURPRISE THIS HEAT
ACTIVATED EXTRACT STILL HAS
ACTIVITY, RESISTANT TO HEAT
TREATMENT, RESIST APT TO PROTEIN
SK, RESISTANT TO NUCLEACES AND
TURNS OUT TO BE A SMALL MOLECULE
EMPLOY AND BASED ON THIS AND
OTHER SPRMS, WE PROPROPOSED THAT
PERHAPS, THE DNA SENSOR IS AN
ENZYME AND THIS ENZYME CAN MAKE
A SMALL MOLECULE THAT THEN
FUNCTIONS AS A SECOND 1 THAT
WOULD BIND AND ACTIVATE TAIN TO
INDUCE IN THE FILMS, AND THIS
MODEL WAS QUITE PROVOCATIVE AT
THE TIME BUT IT TURNED OUT TO BE
CORRECT EMPLOY SO TO MAKE A LONG
STORY SHORT, WE PURIFIED THE
SMALL MOLECULE AND IT TURNS OUT
THAT IT'S A CYCLO DINUCLEOTIDE,
SO THIS IS G-PART, THIS IS AMP
PART, THIS IS 12 IS 5 FRIEM AND
THE OTHER 1 IS 5 PRIME 3 TIME
AMERICA YC EMPLOY SO WE CALL IT
3 PRIME, 2 PRIME CGAMP, SO
THEY--THE KRETICALLY CAN YOU
MAKE 4 ISOMERS.
SO IN ADDITION TO 2 PAM, 2 PAM
CGAMP, OR 3 PAM, 3 PAM, CGAMP
AND WE MAYBE ALL THIS IS MAJOR
TO BYPASSING TO STAIN AND WE
FOUND THAT THIS 2 PAM 3 PAM
CGAMP, THE CANNED WHAT THAT OUR
CELLS MAKE, IT'S ABOUT HIGHER
AFFINITY THAN OTHER CGAMP
ISOMERS AND IF WE DELIVER THESE
2 PAM, 3 PAM CGAMP, IT CAN
INDUCE BETA WITH ABOUT 20 NANO
MOLAR EMPLOY SO CGAMP IS INDEED
A SECURITIZATION. MESSENGER THAT
COMBINE AND ACTIVATE STING TO
INTRODUCE INTERFERON BETA.
NOW THE QUESTION IS WHAT'S THE
ENZYME AND IT TURNS OUT THAT
THIS ENWRIEM WAS VERY DIFFICULT
TO PURIFY.
WE WENT THROUGH MORE THAN 2000
DISHES OF CELLS, THOSE ARE
REALLY LARGE DISHES OF CELLS AND
WE WENT TO DIFFERENT
PURIFICATION STRATEGIES, BUT WE
BEINGN'T PURIFY TO HOW MUCH AN
ENTITY WOULD ACHIEVE ONLY ABOUT
15,000 PURIFICATION AND WE
ESTIMATED THAT WE NEED TO PURIFY
THESE LIKE MEANING TO ACHIEVE
IMIEWPITY, AND THAT WAS VERY
DIFFICULT, AND SO WE WERE STUCK
THERE WE PUT THIS ON THE
COLLAGEN AND THEN IT WOULD
DISAPPEAR AND THEN WE HAVE TO
START OVER.
SO THEN WE CAME UP WITH A
STRATEGIC PLANEMY, WE REASONED
THAT OKAY IF WE HAVE PURIFIED
THIS BY [INDISCERNIBLE], WE HAVE
MUCH TO GO AND THIS IS ABOUT 1%
PURITY AND IF WE IDENTIFY ALL
PROTEINS OUT, 1 OUT OF 100 WOULD
BE OUR PROTEIN OF INTEREST BUT
WE JUST DIDN'T KNOW WHICH 1.
BUT HOWEVER, IF WE USE MASS
SPEC. TO IDENTIFY ALL THE
PROTEIN ANDS DIFFERENT
FRACTIONS, AND QUANTIFY THE
ABUNDANCE, THE ABUNDANCE OF OUR
PROTEIN SHOULD CO PURIFY WITH
OUR ACTIVITY, AND SO, I RUN THIS
IDEA BY MY STUDENT YIERK AXI,
WU, HE LEARNED HOW TO DO MASS
SPEC. AND BECAME VERY GOOD AT IT
AND HE CUT OUT MORE THAN A
HUNDRED BANDS FROM DIFFERENT
PUREICTION STRATEGY ANDS
IDENTIFY AND QUANT PIE THESE
PROTEINS AND HE FOUND THERE WERE
3 PROTEINS THAT CO PURIFIED WITH
ACTIVITY THROUGH DIFFERENT
PURIFICATION STRATEGIES.
IN 1 OF THESE PROTEINS, WAS A
PROTEIN IN A DATABASE WITH A
CHARACTERIZED FUNCTION AND WE
CALL THIS NOW, WE CALL IT CGAS
AND SEQUENCE ANALYSIS SHOWED
THAT THIS PROTEIN HAS A
NUCLEOTIDE TRANSFER ACE DOMAIN
WHICH IS HOMOLOGOUS TO ANOTHER
ENZYME, OS-1 AND OLIGIO SYNTHASE
1 AND THE HIGHLY [INDISCERNIBLE]
ARE HIGHLY CONSERVED.
SO WE PUBLISH 2 PAPERS THAT YEN
JUST MENTIONED AND FOR THOSE WHO
ARE INTERESTED IN HOW WE
DISCOVERED CGAS AND CGAMP, CAN
YOU SCRUTINIZE THESE PAPERS.
OKAY, SO, THAT'S HOW WE
DISCOVERED CGAZ AND CGAMP.
AND NOW FAST FORWARD MANYIERS
LATER WE HAVE A MORE
UNDERSTANDING OF THE CGAS
STAINING PATHWAY AND I WILL USE
THIS CARTOON TO ILLUSTRATE THIS
PATHWAY.
SO HERE IS A CELL, THERE'S
INFECKED BY A VIRUS, IN THIS
CASE, A DNA VIRUS, THE VIRUS
DELIVERED DNA INTO THE CELLS,
AND THE DNA AND THE CYTOPLASM IS
A DENDRITIC CELL ROUGH ATOM
SIGNAL THAT IS RECOGNIZED BY
THIS ENZYME CGAS.
CGAS BINDS TO THE DNA AND IT
BECOMES ACTIVATED.
SO THIS A STRUCTURAL OF CGAS,
WHEN IT'S BOUND TO DNA, SO DNA
BINDING PUSHES THIS PINK
ACTIVATION LOOP AND INDUCES A
CONFIRMATIONAL CHANGE IN THE
ACTIVE SITE OF THE ENSWROIM AND
THIS DNA BINDING ALSO INDUCES
ACTIVATION OF ENZYME WHICH IS
IMPORTANT FOR ACTIVATION.
SO NOW THIS ENZYME IS ACTIVATED
FROM CTP AND ATP INTO THE C-GAS
MOLECULE WHICH ACTIVATES IMMUNE
RESPONSE.
SO NOW 1 MOLECULE OF ENZYME
MAKING MULTIPLE MOLECULES OF
THIS SECOND MESSENGER, SO THE
SIGNAL IS AMPLIFIED.
AND EACH OF THESE SECOND
MESSENGER COMBINES TO A SKIN
DIMER AND STAIN IS NOW ACTIVATED
AND WHAT LEADS TO ACTIVATION OF
NF-KAPPAB AND IR3 WHICH MOVE
INTO THE NUCLEUS TO TURN ON THE
EXPIRATION OF HUNDREDS OF YEENS
THAT ARE IMPORTANT IN IMMUNE AND
INFLAMMATORY RESPONSES.
THIS IS ANOTHER YOU VIDEO OF THE
PATHWAY WHICH IS CGAS ACTIVATING
THE BINDING DNA WHICH WILL MAKE
THE DISCIPLINARY SULFURIDE
NUCLEOTIDE AND ACTIVATES STING
AND ACTIVATES IPPK, AND IRF3,
WHICH THIS WILL BE SHOWN IN THE
FILMS.
I WILL USE THIS SLIDE TO
HIGHLIGHT 1 IMPORTANT POINT AND
THAT IS THAT CGAS IS ACTIVATED
BY ANY DOUBLE STRANDED DNA
INDEPENDENT OF A DECKER NA
SEQUENCE.
AND THE CRYSTAL STRUCTURE OF THE
CGAS DNA COMPLEX FINDS IT
BEANEDS WITH THE SUGAR COMPOUND
AND THAT EXPLAINS WHERE IT'S DMA
SEQUENCE INDEPENDENT.
AND I REALLY LIKE THESE FOR 2
REASONS, 1 IS THAT, WELL, WE
DON'T HAVE TO DO DNA SEQUENCING
SO THERE'S A LOT OF SEQUENCING,
AND ABOUT SECONDARY AND MORE
IMPORTANTLY, THIS PROBABLY
ALLOWS THE SINGLE PROTEIN TO
DETECT A LARGE VARMIS I HAVEETY
OF PATHOIENS AS LONG AS THEE
PATHOIENS CONTAIN DNA OR REQUIRE
DECKER MA IN THE LIFE CYCLE, SO
WE AND OTHERS HAVE SHOWN THAT
CGAS IS INDEED A DNA SENSOR FOR
DNA VIRUSES SUCH AS HE WERE EASE
VIRUS, [INDISCERNIBLE] VIRUSES
SUCH AS HIV, MDB, AND PARASITES
SUCH AS MALARIA.
ANOTHER 1 THAT CAN ACTIVATE CGAS
AND ACTUALLY OUR OWN DNA.
AS I MENTIONED OUR OWN DNA IS
USUALLY IN THE IN YOU KLF-TWOYUS
AND IN THE MITOCHONDRIA BUT
UNDER SOME PATHOLOGICAL CONDIGS,
IF OUR OWN DNA GETS IN THE
CYTOSOL, IT CAN LEAD TO A
VARIETY OF AUTOIMMUNE AND
DISEASES.
AND 1 EXAMPLE OF AUTOIMMUNE
DISEASE IS CALLED
[INDISCERNIBLE] OR IEE, AUDIENCE
GS AND THESE ARE THE CHOSEN WITH
THE DISEASE, THEY HAVE SEVERE
CALCESKS IN THE BRAIN AND THAT'S
BECAUSE WHEN THESE KIDS WERE
BOARN THEY PRODUCE THIS TYPE 1
INTERFERON THAT DAMAGE THE BRAIN
IN THE ABSENCE OF APPEARING
INFECTION ANDS THESE ARE
MONOGENIC DISEASES AND MANY OF
THESE YEENS HAVE BEEN IESHES
DENTIFIED BY [INDISCERNIBLE] AND
OTHERS AND MOST OF THESE GENE
MUTATIONS LEAD TOACIVATION OF
THE CGAS STAINING PATHWAY.
SO FOR EXAMPLE, 25% OF THESE
PATIENTS HAVE MUTATIONS IN THIS
GENE CALLED TRX1 WHOSE ENCODE
NUCLEUS THAT DIGESTS DNA IN THE
CYTOPLASM.
AND SO LOSS OF FUNCTION
MUTATIONS OF THIS GENE CRIPPLE
ACTIVITY OF THE ENZYME AND THIS
ALLOWS CYTOPLASMIC DNA
ACCUMULATE ASK THAT TRIGGERS THE
PRODUCTION.
THE QUESTION IS WHETHER THE KIDS
HAVE THE DISEASE BECAUSE THEY
HAVE HYPER ACTIVATION OF THE
CGAS PATHWAY.
SO TO TEST THESE, WE USE
[INDISCERNIBLE] DEFICIENT MICE.
AND THESE MICE DIE WITHIN A FEW
WEEKS AFTER BIRTH BECAUSE THEY
HAVE VERY SEVERE INFLAMMATION IN
MANY ORGANS AND WE ASKED IF WE
REMOVE CGAS FROM TREX1, KNOCKOUT
MICE, CAN WE RESCUE THE DISEASE
IN SO WE GENERATED TREX1 CGAS
DOUBLE KNOCKOUT MICE AND NOW A
HUNDRED PERCENT OF THE MICE
SURVIVE AND THEY DIDN'T HAVE ANY
SYMPTOMS OF DISEASE EMPLOY
INTERESTINGLY IF WE JUST REMOVE
1 CGAS, WE CAN LARGELY RESCUE
SURVIVAL AS YOU CAN SEE HERE.
SOPHISTICATEDY WE THINK THAT
THIS HAPLOINSUFFICIENCY OF CGAS
IS DUE TO THIS PHENOMENON THAT
WE DISCOVERED RECENTLY CALLED
BASICALLY IT'S A DNA INDUCED
[INDISCERNIBLE] SEPARATION OR
CGAS.
SO BASICALLY CGAS HAS MULTIPLE
DNA BINDING DOMAINS AND THEY
COMBINE TO DECKER NA TO
MULTIVARIANT INTERACTIONS AND
THIS MULTIVARIANT INTERACTIONS
BETWEEN DNA AND CGAS, DRIVE THE
SEPARATION THAT LEAD TO
FORMATION OF THESE BASE IN WHICH
CGAS IS CONCENTRATED AND
ACTIVATED.
AND WE THINK THAT THIS LIQUID
PHASE SEPARATION OF CGAS RENDERS
THIS ENZYME HIGHLY SENSITIVE TO
THE CYTOPLASMIC CONCENTRATION OF
BOG DNA AND CGAS.
SO, FOR EXAMPLE, IN TREX 1
DEFICIENCY CELLS, CYTOPLASMIC
DNA IS ELEVATED BUT STILL QUITE
LOW.
SO THESE THEN, IF WE BRING THE
CGAS CONCENTRATION DOWN BY 50%,
THAT BRINGS IT DOWN FOR THE
SEPARATION AND SO CGAS, IF YOU
HAVE 50% OF CGAS, IT CANNOT
SEPARATE AND CANNOT SEPARATE TO
STOP THE DISEASE SO WE THINK
THIS CONTAINS
HAPLOINSUFFICIENCY.
WE THIS IS VERY EXCITING BECAUSE
THIS RESULT AND OTHER RESULTS
THAT I DON'T HAVE TIME TO SHOW
HERE, BASICALLY PROVIDE THE
PROOF OF CONCEPT FOR TARGETING
CGAS.
SO IF WE CAN FOR INHIBITOR OF
CGAS.
THAT EVEN PARTIAL INHIBITION OF
CGAS MAY PROVIDE BENEFITS TO
PASHTS WITH AUTOIMMUNITY
DISEASES SUCH AS ATS, SO MANY
MAPS AND COMPANIES ARE LOOKING
ON THE CGAS INHIBITORS.
SO IN THE PAST 20 YEARS OR SO,
WORK FROM MANY LABS THROUGHOUT
OUR WORK HAS SHOWN THAT THIS
CGAS THIN PATHWAY PLAYS A
CENTRAL ROLE IN MANY
PHYSIOLOGICAL PATHOLOGICAL
PROCESSES.
AND THERE ARE TOO MANY PAPERS IN
IN SPACE, YESTERDAY, I CHECKED
PUB MED, DID A SEARCH WITH CGAS
AND STING AS KEY WORDS AND IT
RETURNED MORE THAN 2000 PAPERS
SINCE 2013 AND THIS YEAR SO FAR,
THERE WERE 480 PAPERS WHICH
MEANS EVERY DAY, YOU HAVE ALMOST
2 PAPERS ON THIS PATHWAY WHICH
IS DIFFICULT TO CATCH UP, SO
HERE I WANT TO SUMMARIZE, YOU
KNOW TRY MY BEST TO SUMMARIZE
THIS WORK AND IN THIS SLIDE.
SO ACTIVATION IMPOSES VIRUSES,
PARASITES, ACTIVATION ON THIS
PATHWAY CAN LEAD TO A VARIETY OF
AUTOIMMUNE AND INFAMOUS DISEASES
SUCH AS AGS, SAVI, AS YOU KNOW
FROM DR. ELA'S WORK THIS HAS
BEEN SHOWN TO LEAD TO
NEURODEGENERATION VERY NICE WORK
FROM HITCH ARD YU HAS SHOWN HERE
THAT AND PATH THIS PATHWAY CAN
LEAD TO NONKAN--KANA CANONICAL
FORM OF AUTOPHAGY AND WE AS MANY
AS OTHER LABS HAVE SHOWN THAT
ACTIVATION ON THIS PATHWAY CAN
LEAD TO CELLULAR SENESCENSEL ASK
THIS PATHWAY IS ALSO VERY
IMPORTANT FOR IMPACT HUMAN
IMMUNITY.
SO THAT'S OUR INTRODUCTION AND
NOW I WILL TALK ABOUT THE
REGULATION OF CGAS SIGNALING IN
ANIMAL CELLS EMPLOY SO TO THIS
SLIDE, SO CGAS IS PRODUCED BY
CGAS, THE QUESTION IS HOW DID
THAT ACTIVATE THE PATHWAY
THROUGH STING AND SPECIFICALLY
WE ASK HOW DOES CGAS ACTIVATE
STING, HOW DOES STINTH ACTIVATE
CP1, AND WE STARTED THIS FIRST
BECAUSE OF THE EXPERIMENT WE DID
MANY YEARS AGO.
SO HERE WE CAN SEE
PHOSPHORYLATION AS WELL AS
PHOSPHORYLATION OF 3, AND THAT
IS EXPECTED BECAUSE THE TPK1 IS
THE KINASE PHOSPHOR IL3, IF WE
STIMULATE CELLS WITH DNA OR THE
GENOMIC PATHWAY, WE CAN ALSO SEE
TBK ACTIVATION AS WELL AS WELL
AS PHOSPHORYLATION OF IRF3.
NOW WE CAN SEE TPK 1 ACTIVATION,
BUT WE COULDN'T SEE IERK L3
PHOSPHORYLATION EMPLOY AND
THAT'S SOMEWHAT STRANGE BECAUSE
IT IS VERY WELL KNOWN THAT TPK1
IS THE KINASE, AND SO WHY IT
THAT WE CAN SEE ACTIVATIONOT
KINASE BUT NOT FORFORALATION OF
THE SUBSTRATE.
AND THE SIMPLEST MODEL THAT
COULD EXPLAIN THIS RESULT IS
THAT TPK1 IS NECESSARY BUT NOT
SUFFICIENT TO ACTIVATE IL3.
SO HERE WITHY IF WE LOOK AT 3
DIFFERENT PATHWAYS THAT CAN LEAD
TO THE ACTIVATION OF TPK1 AND
ICASHING K, SO THIS IS THE RNA
PATHWAY THAT ACTIVATE THESE IN
MAPS, THIS IS THE DNA PATHWAY TO
ACTIVATE STAIN AND THIS IS IERK
L1 PATHWAY THAT ACTIVATE THIS
PROTEIN CALLED [INDISCERNIBLE] 6
AND WE SHOWED THAT IT IS
UBIQUITIN E3 LIGASE THAT
CHARACTERIZE THE POLYUCIBOL
WINNATION THAT LEADS TO
ACTIVATION OF IKK.
AND TARE, AF6 CAN ALSO ACTIVATE
TBK1, IN CONTRAST, TBK IS
ACTIVATED BY STING OR MAVS, SO
THERE MUST BE SOMETHING SPECIAL
ABOUT MAVs ANDS STING AND
THEIR ABILITY TO NOT ONLY
ACTIVATE TPK1 BUT ALSO TBK1 TO
MAKE IERK RF3.
AND THIS WAS PERFOR THE PURPOSED
BY A POST DOC STUDENT AND AT THE
TIME THIS OPPORTUNITY WAS
WORKING ON REGULATION OF MAVS BY
RNA RIEROUS INFECTION AND SHE
FOUND THAT MAVS IS
PHOSPHORYLATED SHOWN HERE IN
RESPONSE TO THE ACTIVATION, SO
SHE FOUND THIS SHORT SEQUENCE
MOTIF THAT IS HIGHLY CONSERVED
NOT ONLY IN MAVS BUT ALSO IN IL3
AND STING.
SO THE PHOSPHORYLATION OF IL3 OF
THIS RESIDUE DUE TO DIMERIZATION
AND ACTIVATION, AND SO SHE ALSO
SHOWED THAT STING IS INDEED
PHOSPHORYLATED AND
[INDISCERNIBLE] AT THE
C-TERMINAL TAIL OF STING.
AND SHE BASICALLY USE HER EYES,
NOT COMPUTER TO IDENTIFY AND
SHOW SEQUENCE MOTIFS WHICH WAS
REMARKABLE AND THIS SEQUENCE
MOTIF ACTUALLY CONSERVING OTHER
PROTEINS INROFULLED IN INNATE
IMMUNITY FOR EXAMPLE IS ALSO
FOUND IN THIS ASH DATA
PROTECTIONER PROTEIN CALLED TRIP
WHICH IS INVOLVED IN TLR4
SIGNALING.
SO WHEN IS COMMON AMONG ALL
PATHWAYS CAN LEAD TO INDUCTION
FOR ALL INTERFERONS, SO MAVS,
STING AND TRF, THEY CALL WHAT WE
CALL A CANNED WHAT MOTIF, AND SO
WHEN EACH OF THESE RECEPTORS IS
ENGAGED BY AN UPSTREAM LIGAND,
IT WOULD LEAD TOACIVATION OF THE
KINASE IPK AND ITISSUING T1,
WHICH WOULD SEQUENCE THE MOTIF,
SO NOW THE ADAPTER PROTEIN IS
PHOSPHORYLATED AND THE IL3,
BECAUSE IL3 CONTAINS THIS
POSITIVELY CHARGED SURFACE, THAT
CAN BIND TO THIS PHOSPHORYLATED
MOTIF, THEN THIS, FOR THE
PHOSPHORYLATION BY TPK1, NOW
IL3, IS PHOSPHORYLATED, IT FORMS
A DIMER AND MOVES INTO A NUCLEUS
TO INDUCE TYPE 1 INTERFERONS SO
WHAT WE'RE ROUGH ATOM POSING
HERE IS THAT FORFORALATION OF
THIS ADAPTER PROTEINS, SEQUENCE
MOTIFS, PRORIDES LICENSING
MECHANISM THAT ARK LOWS THE
KINASE TO PHOSPHORYLATE THE
SUBSTRATE AND FOR IRPT LUKEIN 1
AND TNF, THAT DON'T ENGAGE ANY
OF THESE ADAPTIVE PROTEINS, AND
THAT'S WHY THEY CAN LEAD TO IL3
ACTIVATION WAS FOR THIS OTHER
PATHWAYS THAT ENGAGE THESE
ADAPTER POLINGS THAT CONDUCT THE
IL3 AND WE THINK PROVIES A
UNIFIED MECHANISM THAT EXPLAINS
WHY THESE PATHWAYS THAT WE
PRESENT VIRUS INFECTION AND
OTHER INFECTION CAN LEAD TO TYPE
1 INTERFERON PRODUCTION.
HOWEVER FOR EXPERTS IN INNATE
IMMUNITY HERE, WHAT SAY OKAY,
WHAT ABOUT A MECHANISM?
WHAT ABOUT A TOLL-LIKE RECEPTOR
7, 8, AND 9 WHICH CAN ALSO
INDUCE TYPE 1 INTERFERONS?
AND THIS QUESTION WAS ADDRESSED
BY THIS VERY NICE PAPER FROM
JULIA [INDISCERNIBLE] ABOUT 3
YEARS AGO AND THEY IESHES
DENTIFY A PROTEIN CALLED TESLA
AND A FUNCTION IN THE
ENDOSTUDIES OF MULTIPLE
ENDOCRINAL RECEPTOR PATHWAYS SO
FOR THESE RECEPTORS, TLR7, 8,
AND 9 THEY DID TECT RNA OR DMA
LIGANDS AND THEN, THEY WERE
ACTIVATED AT THE MYD88 PATHWAY
AND ACTIVATION OF IKKBETTA IN
THIS CASE WERE PHOSPHORYLATE
THIS TRANSLATION FACTOR IRF5
WHICH WOULD LEAD TO INDUCTION OF
TYPE 1 ISHT FERON AND OTHER
INFLAMMATORY CYTOKINES.
HOWEVER, THE KEY POINT HERE IS
THAT IN ORDER FOR IKKF BETA TO
PHOSPHORYLATE IL5, IT MUST FIRST
PHOSPHORYLATE THE PROTEIN THAT
ALSO HAS THIS PLACE MOTIF THAT
WE IDENTIFY SO ONLY AFTER THIS
PLACE MOTIF IS CO REG IEWLGTED
CAN WE GET IKKB BETA SO THE
MECHANISM IS SIM ARRA TO CGAS
STAIN PATHWAY.
THE MAVS AND STING PATHWAY.
SO NOW BACK TO THE STING
PATHWAY.
SO NOW WE HAVE A BETTER
UNDERSTANDING OF HOW TPK1
ACTIVATE IL3, SO THE BOTTOM LINE
HERE IS THAT IN ORDER FOR TPK1
TO PHOSPHORYLATE 3, IT MUST
FIRST PHOSPHORYLATE THE ADAPTING
STAIN.
SO WE JUST ADD ANOTHER QUESTION
TO THE LIST AND TO BE ABLE TO
ANSWER ALL THESE QUESTIONS WE
FEEL THAT WE NEED TO SOLVE THE
STRUCTURE OF THE FOOD AND STAIN
PROTEIN.
SO STAIN IS A SMALL
TRANSMEMBRANE PROTEIN, IT'S
ABOUT 40 KILODALT OPEN MEETING
AND A 4 TRANSMEMBRANE DOMAINS
AND THE ER MEMBRANE AND HAS THE
LIGAND BINDING DOMAIN THAT BIENS
TO CGAMP AND IT CONTAINS THIS
RESDUE THAT GERONTOLOGYSTSS
FOSTER NURSED FOCUSED ON
FORALATED.
AND PRIOR TO THIS WORK THE
BINDING STRUCTURE OF THE LIGAND
DOMAIN STAINING BUT WE DIDN'T
HAVE THE STRUCTURAL INFORMATION
FOR THE REST OF THE MOLECULES.
SO WE ASSEMBLE A TEAM TO TRY TO
SOLVE A STRUCKURE OF THE FOOD
AND STAIN, THIS A POST DOC IN
THE PROFESSOR'S LAB AND WE'RE
FORTUNATE TO COLLABORATE WITH A
CRYOEM EXPERT [INDISCERNIBLE]
AND TOGETHER THEY SOLVE THE
STRUCTURE OF FULL LENGTH STING
WHICH I THINK IS QUITE
REMARKABLE BECAUSE STING IS A
SMALL MEMBRANE PROTEIN, EVEN A
SKIN DIMER IS ONLY 80 DILL O
DALTON WHICH IS SMALL FOR CRYO
EM.
SO IN ANY CASE, SOPHISTICATED
SOLVE THE STRUCTURAL OF FOOD AND
STING PROTEIN, SO STING FORMS
THE SUBDIMER AND THE MONOMERHAS
4 TRANSMEMBRANE HELICS AND
CONNECKS TO THE DISCIPLINARY
LAND BINDING DOMAIN AND THIS
BINDING DOMAIN FORMS THIS CGAMP
BINDING POCKET.
SO THIS IS THE STRUCTURE OF
STING IN THE ABSENCE OF THE
LIGAND TO PERFORM THE STING.
SO WHAT HAPPENS IF YOU ADD
LIGAND TO THE STING, SO STING
WILL COME IN HERE AND BIND HERE
AND INDUCES 1 ACHE DEGREE
ROTATION OF THE LIGAND BINDING
DOMAIN RELATIVE TO THE
TRANSMEMBRANE DOMAIN AND THE
CGAMP INDUCES CONFIRMATIONAL
CHANGE SUCH THAT STRUCTURAL COME
CLOSER TO EACH OTHER AND NOW WE
CAN SEE THIS NEW STRUCTURE.
SO NOW IF WE LOOK AT THE
STRUCTURE OF STING, AND THE
PRESENCE OF THE LIGAND OR IN THE
ABSENCE OF LIGAND AND COMPARE
THIS STRUCTURES CAN WE NOTICE
SOME INTERESTING FEATURES, SO
NOW IF WE ZOOM IN TO THIS
REGION, AND SO THIS--THIS IS A
STRUCTURAL OF STING BOUND TO
CGAMP AND YOU CAN SEE THAT THE
PROHELIXES ARE FALLING APART AS
COMPARED TO THE [INDISCERNIBLE]
PERFORM OF STING.
SO THIS THESE 2 HELIXES ARE SO
CLOSE TO EACH OTHER THAT THEY
CAN ACCOMMODATE ONLY A SMALL
AMINO ACIDS SUCH AS A
[INDISCERNIBLE].
SO A PREDICTION IS THAT IF YOU
CHANGE THIS TO A LARGER RESIDUE,
IT MIGHT INDUCE SPONTANEOUS
ACTIVATION WITH STING.
AND THAT'S INDEED THE CASE, SO
HERE IF WE CHANGE THIS TO AMINO
ARK SIGNIFYS WE CAN SEE
INDUCTION OF BETA, IN ABSENCE OF
STING, WHERE THE WILD-TYPE STING
ACTIVATION, INDUCTION OF
INTERFERON ABOUTETTA DEPENDS ON
CGAMP.
AND WE CAN ALSO SEE THIS BY
LOOKING AT THE SIGNALING CASCADE
AND SEE ACTIVATION OF TBK IL13,
FOR THIS IN THE CELLS THAT CAN
BE THE MUTANTS, EVEN IN THE
ABSENCE OF CGAMP, WHEREAS FOR
THE WILD-TYPE STING THAT IT
REQUIRES THE PRESENCE OF CGAMP.
THIS IS VERY INTERESTING BECAUSE
MUTATIONS OF THESE RESIDUES HAVE
BEEN FOUND IN HUMAN PATIENTS
WITH THIS AUTOINPOLICEMANNATORY
DISEASE CALLED SAVI, OR SUSTAIN
[INDISCERNIBLE] VASC LOP ALGTY
ALMOST IN EARLY INFANCY AND THE
SARI DISEASE WAS NICELY
CHARACTERIZED BY RAFAEL
[INDISCERNIBLE] HERE AT NIH.
SO SPONTANEOUS ACTIVATION OF
STING WITH LEAD TO SEVERE CASES.
OKAY, AND SO WE ALSO SOLVE THE
STRUCTURAL STING BOUND TO TPK1
WHICH WAS PUBLISH INDEED 1 OR
WOPAPERS AND HERE I WOULD JUST
SUMMARIZE THE MODEL BASED ON
THESE STUDIES.
SO HERE IS A STING DIMER THAT IS
ON THE ER MEMBRANE AND THIS IS
THE CGAMP MOLECULE IN RESPONSE
TO DNA, SO THIS CGAMP MOLECULE
BINDS TO STAIN DIMER AND INDUCES
180-DEGREE ROTATION, OR THE
LIGAND BINDING DOMAIN, WITH THE
TRANSPLANT, RELATIVE TO THE
TRANSMEMBRANE DOMAIN, AND THIS
CGAMP BINDING INUS DOOS
CONFIRMATIONAL CHANGE SUCH THAT
IT CAN FORM THE OLIGMER.
AND THIS USES THE C-TERMINAL
CARE TO RECRUIT THE KINASE TPK1
AND AS A RESULT OF THIS
AUTOMATIZATION, IT BRINGS THE
TP1 KINASE CLOSE TO EACH OTHER
SO THEY COME CLOSE TO
PHOSPHORYLATE ITSELF AND THAT'S
FOR THE TPK1 ACTIVATION.
NOW THAT THIS ACTIVATED KINASE
WILL INVOLVE THE TAIL THAT IS
NOT USED FOR RECRUITMENT, SO
THIS STAINED CTL, IS
PHOSPHORYLATED AND ANOTHER STING
TAIL IS RECRUITED AND
PHOSPHORYLATED AND THE
FORFORALATED SEE THE TERMINUS
TAIL CAN THEN RECRUIT THIS
TRANSCRIPTION FACTOR IL3 IN
POSITION, THE RESIDUE OF IR3 FOR
TPK1 AND SO NOW IL3 AND
PHOSPHORYLATED AND ANOTHER IL3
AND RECRUITED, AND
PHOSPHORYLATED AND
PHOSPHORYLATED IL3 FORMS A
DIMER, MOVE INTO THE MUSEUM
CLEUS TO INDUCE TYPE 1
INTERFERONS.
SO THIS IS A MODEL BASED ON
STRUCTURE AND BIOCHEMICAL DATA
AND THE QUESTION IS WHETHER IT'S
TRUE IN VIVO, AND SO WE
DPENERATED STAINED KNOCK-IN MICE
USING CRSPR, TO MAKE MORE MUSEUM
TAIGS WITH THE STAINED TAIL THAT
ABROGATE AND BINDING TO TPK1 OR
BINDING TO IL3 AND WE SHOW THAT
BASICALLY THIS MODEL AND
SPECIFICALLY WE FOUND THAT TPK1
RECRUITMENT TO STAIN IS VERY
IMPORTANT FOR ACTIVATION OF BOTH
IERK L3 AND THE NFCAPPA AND
THAT'S IMPORTANT FOR ANTITUMOR
IMMUNITIASMUNE DEFENSE AGAINST
VIRAL INFECTION, IN THIS TPK1
RECRUITMENT FOR STAIN SUGGEST
ALSO VERY IMPORTANT FOR
AUTOIMMUNE DISEASES CAUSED BY
DNA EMPLOY OKAY, SO IN THE
REMAINING FEW MINUTES, I WILL
TURN THE CLOCK BACK BY
[INDISCERNIBLE] AND TALK ABOUT
BACTERIAL CGAS AND IMPACT FOR
IMMUNE DEFENSE.
SO THROUGHOUT BILLIONS OF YEARS
OF EVOLUTION, BASICALLY THIS
CONSTANT ARMS RACE BETWEEN
BACTERIA AND BACTERIAL PHAGES
AND THAT GIVES ENORMOUS
DIVERSITY AND COMP A XATOXITY
FOR BACTERIAL PHAGES.
IT IS ESTIMATED THAT THERE ARE
10-30 FAINLS ON A PANEL AND THAT
IS MORE THAN OTHER ORGANISMS
COMBINED INCLUDING BACTERIA.
LIKE ANY VIRUSES, BACTERIAL
PHAGES THAT HAVE DNA OR RNA
DENOAMS WOULD A DOUBLE STRANDED
DNA GERONTOLOGYSTS NEMS COMPRISE
IN MORE THAN 95% OF BACTERIAL
PHAGES AND AS I MENTIONED
EARLIER BACTERIAL HAS POWERFUL
AND SOPHISTICATED IMMUNE SYSTEMS
SUCH AS RESTRICTIONS USING
CRSPR, AND 1 IMPORTANT
DEVELOPMENT IN THE CGAS STAIN
FIELD IS THE DORPHY OF A LARGE
FAMILY OF CGAS LIKE ENZYMES IN
BACTERIA.
SO PHILLIP'S [INDISCERNIBLE] LAB
AT HARVARD MEDICAL SCHOOL IERK
DENTIFY ABOUT 6000 BACTERIA WILL
CGAS LIKE PROTEINS IN VARIOUS
BACTERIAL.
WORK FROM [INDISCERNIBLE] ISRAEL
AND PHILLIP [INDISCERNIBLE] HAS
SHOWN THAT THE MAJOR FUNCTION OF
THIS BACTERIAL CEMBASSY LIKE
ENZYMES IS FOR IMMUNE
DIFFERENCE, FOR EXAMPLE IN THIS
PAPER, FROM THE LAB ISSUE THEY
SHOW THAT THIS BACTERIAL CGAS
ENZYME IS ACTIVATED BY PHAGE
INFECTION TO AN UNKNOWN
MECHANISM AND THIS ENZYME THEN
CONVERTS TO GTP AND ATP TO THE
MOLECULE WHICH IN THIS CASE WERE
BACTERIAL [INDISCERNIBLE] AND
THIS LIPASE HAS THE MEMBRANE
LIPID AND CAUSES THE VIRUS
INFECTOR CELLS TO COMMIT SUICIDE
AND THIS IS THE MECHANISM TO
LIMIT THE VIRUS PREDOMINANTLY TO
PROTECT THE BACTERIAL COMMUNITY.
SO THIS CGAS NEW DEFENSE SIZE IS
ALSO CALLED CBASS, OR CYCLIC
OLIGONUCLEOTIDE-BASED ANTIPAGE
SIGNALING SYSTEMS, SO THIS CGAS
IS THE GENE IS QUITE OFTEN NEXT
TO THE GENE ENCODING THE
EFFECTOR PROTEIN SUCH AS A
PHOSPHOR LIPASE.
AND INTERESTINGLY, ABOUT 40% OF
THIS CBASS SYSTEM WHICH MORE
THAN 2000 CBASS SYSTEM HAVE
GENES THAT ENCODE ENZYMATIC
DOMAINS THAT UBIQUITIN E1 OR E2
ENZYME DOMAINS AS WELL AS
[INDISCERNIBLE] ENZYME DOMAINS.
I WAS REALLY INTRIGUED BY THESE
FINDINGS BECAUSE AS I MENTIONED
IN MY PREVENTIVUOUS LIFE I WAS
WORKING ON UBIQUITIN SYSTEM.
SO AT THIS TIME, POST DOC JOINS
THE LAB AND 1 OF THE FIRST
QUESTIONS THAT JUSTIN ASKED IS
WHETHER THE BACTERIAL E1, E2
PROTEINS CONFIRM WITH A
BACTERIAL PROTEIN MUCH LIKE
UBIQUITIN IN THE MAMMALIAN
SYSTEM.
SO HE FOCUSED ON THIS SO CALLED
CAP 2 PROTEIN WHICH IS AN FUSION
PROTEIN EMPLOY SO HE TOOK THIS
CGAS FROM THIS STRAIN OF E.COLI
AND TRANSFERRED IT TO THE
LABRATORY TRAIN OF E.COLI WHICH
DOESN'T HAVE THE CBASS SYSTEM
AND HE EPITOPE CAP 2 TO SEE IF
THIS WOULD [INDISCERNIBLE] WITH
THE BACTERIAL PROTEIN AND INDEED
HE FOUND THIS [INDISCERNIBLE]
WITH 50 KILODALTON PROTEIN AND
THIS IS [INDISCERNIBLE] BECAUSE
IT'S SENSITIVE TO DTISSUING
T AND HYDROXYL AMIN THAT IS
INDICATING IT'S CANNED WHAT.
HE THEN CUT OUT THE BAND FOR
MASS SPEC AND TURNS OUT TO BE
BACTERIAL CGAS OR DNCB, IN OTHER
WORDS IF BEHAVIOR YELL CGAS
BEHAVES LIKE UBIQUITIN WHICH IS
VERY STRANGE.
SO TO TEST IF CGAS CONYUIGATES
LIKE UBIQUITIN, JUSTIN DID A
WEST OTHER THAN BLOCK AND HE
FOUND THAT CGAS IN A WILD-TYPE
THAT THE CBASS SYSTEM THAT IT
CONFORMED THIS CONYUIGATE AND
THE FORMATION OF THESE
CONJUGATES DEPENDS ON THE E1 E2
ACTIVITY OF THIS CAP 2 BECAUSE
IF HE MUTATED THE ACTIVE SYSTEM
OF E1 OR E2, THEN WE DON'T SEE
THIS CONYUIGATE ANYMORE.
AND IMPORTANTLY, THE CONJUGATE
FORMATION ALSO DEPENDS ON THE
C-TERMINAL [INDISCERNIBLE] OF
CGAS IF WE CHANGE
[INDISCERNIBLE] TO ALA19 WE
DON'T SHE THE CHAIMPLE THIS THE
CONJUGATES.
AND THE MUTATION OF THIS SITE
RESDUE, THESE 2 ARE SLIGHT
INCREASE OF THESE CONYUIGATES.
SO THE BACTERIAL CGAS INDEED
BEHAVES LIKE UBIQUITIN IN THAT
IT CAN BE ACTIVATED BY E1 AND
EWOAND CONJUGATED TO OTHER
PROTEINS.
THIS IS QUITE STRANGE BECAUSE
THE BACTERIAL CGAS DOES NOT DO
UBIQUITIN AT ALL.
IN PARTICULAR, UBIQUITIN HAS
ALLY CONSERVED HELP THAT HAS 2
[INDISCERNIBLE] TUBES.
FOR BACTERIAL CGAS THEY DON'T
HAVE THESE RESIDUES AND INSTEAD
FOR THOSE CGAS [INDISCERNIBLE]
THEY HAVE THE E1, E2 AND THE
[INDISCERNIBLE] DOMAIN, THEY'RE
HIGHLY [INDISCERNIBLE] IN THE
ALA19, AND FOR THIS PROBLEM THEY
DON'T HAVE THE E1, E2
STRUCTURES, THAT IS NOT
CONSERVED EMPLOY SO JUSTIN
MUTATED THE C-TERMINAL
[INDISCERNIBLE] OF THESE
BACTERIAL CGAS INTO OTHER AMINO
ARK SIDES AND LOOK AT CONJUGATE
FORMATION WITH THE ACCEPTION OR
THIS TO ALA19 SUBTUITION WHICH
CAN STILL FORM CONYUIGATES OR
OTHER AMINO SUBSTITUTION UPON
THE FORMULATION OF THESE
CONJUGATES.
SO CGAS FORMS THIS CONJUGATES TO
CONSERVE THE RESDUE.
NOW THE IMPORTANT QUESTION IS
WHETHER THIS UBIQUITIN CONYU
IMAIGZ IS IMPORTANT FOR IMPACT
PAGE IMMUNE DEFENSE, SO HERE THE
NUMBER OF PHAGE INFECTION,
E.COLI STRAINS, THAT EITHER HAVE
FUNCTIONAL CBASS OR EMPTY
[INDISCERNIBLE].
AND THE FUNCTIONAL CBASS SYSTEM,
REGIEWS THE VIRAL TIGHTER BY
MORE THAN 10,000 FOLD AND THIS
ACTIVITY DEPENDS ON THE PHOSPHOR
LIPASE ACTIVITY OF THE
[INDISCERNIBLE] AND DEPENDS ON
CGAS, DEPENDS ON E1 E2 ACTIVITY
OF CAP 2 AND IMPORTANTLY REMOVAL
OF THE C-TERMINAL GUISEIN AND
C-GAS AND THE EFFECTOR OF THE E1
AND 2 MUTATION AND DISGUISING TO
THIS MUTATION HAS A PARTIAL
PHENOTYPE BECAUSE THIS MUTANT
CAN STILL FORM SOME CONYUIGATES.
SO, SIMILARLY, THE C-TERMINAL
GUISEIN OF C-GAS, PHENOCAPY, E2
E1 MUTATION IN THE CBASS SYSTEM.
SO THIS CGAS CONJUGATION IS VERY
IMPORTANT FOR ANTIPHAGE IMMUNE
DEFENSE.
AND THE QUESTION IS HOW DOES IT
DO THAT?
SO IN THIS EXPERIMENT, JUSTIN
TOOK E.COLI STRAINS THAT HAVE
THE WILD-TYPE CBASS SYSTEM OR
CBASS SYSTEM IN WHICH CGAS
CANNOT BE CONJUGATED.
HE INFECTED THE E.COLI WITH
BACTERIAL PHAGE AND TO MAJOR
CGAMP PRODUCTION, SECOND MEASURE
IN E.COLI AFTER PHAGE INFECTION,
SO CAN YOU SEE THAT FOR THE
WILD-TYPE CBASS SYSTEM, SHOWN IN
RED HERE THEY CAN PRODUCE A LOT
OF CGAMP AND THE CGAMP
PRODUCTION IS REDUCED IN THE
E.COLI STRAIN THAT CANNOT MAKE
OR CONYUIGATE CGAS.
SO MAJOR EECT OF THIS
CONJUGATION WITH CGAS IS TO
GREATLY ENHANCE THIS ACTIVITY
PRODUCE CGAMP.
NOW THE OBVIOUS QUESTION IS WHAT
ARE THE TARGETS OF THIS
UBIQUITIN CONYUIGATION BY CGAS,
SO THEN JUSTIN DID AN
ALTERNATIVE OPROACH WHICH IS THE
FORWARD GENETIC SCREEN FOR THE
ANTAGONISTS OF CGAS CONJUGATION,
SO HERE IF WE INFECT E.COLI WITH
T4 PHAGE, THOSE E.COLI THAT
DON'T HAVE CBASS SYSTEM, THEY
WILL BE INFEBBED BY THE PHAGE,
AND FOR THOSE E.COLI THAT HAVE
THE CBASS SYSTEM THERE WILL BE
ADDITIONAL INFECTION AND FOR
THOSE THAT HAVE THE CBASS SYSTEM
THAT HAVE MUTATIONS IN THE E1 E2
ACTIVITY THEN THEY LOST THE
PROTECTION AGAINST INFEC.
SO JUSTIN MUSEUM TANNIZED THOSE
TO INFECT MUTANTS AND NOW WE CAN
INFECT THE MUTANT CBASS AND SO
PRESUMABLY THERE ARE MUTATIONS
IN THIS T4 PAGES AND SOME OF
THOSE MUTANTS MOOIT ENCODAN
TAGANIST OR CGAS CONJUGATION, SO
THIS SCREEN IDENTIFY AND
CHARACTERIZE 10 KILODALTOR
CALLED BS4.
AND INTERESTINGLY THIS COMBINED
WITH ABOUT 30 NANO MOLAR AND HE
WEPT ON TO SOLVE THE CRYSTAL
STRUCTURE OF BS4 BOUND TO CGAMP
AND HE FOUND THAT BSFORM,
HORMONE LEVELSS A HEXAMERTHAT
BIENS TO 3 MONITOR MERS OF
CGAMP, AND IT'S COMPOSED OF 2
BVS4 MONITOR MERS, SO THESE ARE
THE MODELS.
SO TO VALIDATE THE MODEL JUSTIN
DID INVIF ROW CAP B ACTIVITY, SO
THIS IS A LIGASE ACTIVATED BY
CGAMP AND IF YOU USE THE
WILD-TYPE VIRAL PROTEIN VS4, YOU
IT CAN INHIBIT ACTIVATION BY
CGAMP, AND IF YOU ACTIVATE THE
RESDUES ARE INVOLVED IN THE
OPTIMIZATION OF BS4, THEN THEY
THEY LOST INHIBITING CAP B ISSUE
AND JUSTIN ALSO DID A
BIOINFORMATIC ANALYSIS TO
IDENTIFY VS4 LIKE PROTEINS IN
THE BACTERIAL PHAGE WOULD AND HE
FOUND ABOUT 200 VS4 FLIEK O
PROTEINS AND VARIOUS TYPES OF
PHAGES AND IMPORTANTLY, THE
RESIDUES THAT ARE INVOLVED IN
CGASM BINDING ARE HIGHLY
CONSERVED.
SO BASED ON THESE AND OTHER
RESULTS SO IN RESPONSE TO
INFECTION, FOR THE BACTERIAL
COMMUNITY.
HOWEVER SOME BACTERIAL PHAGES
WILL FIGHT BACK BY ENCODING THIS
VS 4 PROTEIN FUNCTIONS LIKE A
SPONGE, TO SOAK UP THIS CGAMP
MOLECULE AND THAT CAN PREVENT
ACTIVATION AND THAT ALLOWS THE
VIRUS TO CONTINUE TO FINISH THE
VIRAL REPLICATION CYCLE AND
ALLOWS THE VIRUS TO SPRAY.
SO IN IN CASE, THE VIRUS WON.
AND THAT PRODUCES SO MUCH CGAMP
THAT OVERWHELM THE VIRUS AND
THAT WILL ATACT AND KILL THE
VIRUS CELLS, SO THIS IS LIKE A
TYPICAL ARMS RACE IS LIKE A ROCK
PAPER, SCISSOR TYPE OF ARMS RACE
BETWEEN BACTERIA AND PHAGES AND
WE THINK THAT AND WE THINK THAT
THE SYSTEM MIGHT EVOLVE AS A
RESULT OF THIS ARMS RACE BETWEEN
BACTERIA AND PHAGES.
SO WE PUBLISH THIS WORK AND WE
FOUND NOT A GROUP.
THAT AARON WILY, BASICALLY
REACHES THE SAME CONCLUSION FOR
THIS BY CGAS IS IMPORTANT FOR
IMMUNE DEFENSE AND SO ANOTHER
GROUP THAT BY AT UCSF, THEY
IDENTIFY THE SAME RIERALLAN
TAGANIST, FOR THIS, WHICH THEY
CALL ACB 2 THAT FUNCTIONS
THROUGH THIS SPONGE LIKE
MECHANISM TO SOAK UP CGAMP.
SO THIS IS STILL A WORK IN
PROGRESS, FOR EXAMPLE, HOW DOES
CGAS INHIBIT'MUNE FUNCTION
THERE'S NO EVIDENT THAT THE
BACTERIAL CGAS CAN BE ACTIVATE
BIDE DNA ALONE, SO EXACTLY HUGH
IT IS ACTIVATE SIDE STILL NOT
CLEAR.
WHAT ARE THE TARGETS OF CGAS
CONJUGATION, THESE TARGETS COULD
BE PROTEINS, COULD BE LIPIDS
MUCH LIKE AUTOPHAGE EXPE STILL
DO REMAINS TO BE DETERMINED.
HOW DOES CGAS CONYUIGATION
ACTIVATE THE ANTIPHAGE IMMUNITY
AND THE OTHER TARGETS OF THAT
CONJUGATION ENSWROIMS IN
BACTERIA.
SO THERE ARE STILL MANY
QUESTIONS TO ANSWER AND I THINK
THIS IS A RAPIDLY ADVANCING
FIELD.
OKAY, SO IN CLOSING, I JUST WANT
TO HIGHLIGHT THE FACT THAT CGAS
JOINS ENZYMES AND CRSPR AS
IMPORTANT IMMUNE DEFENSE
MECHANISM.
IN BACTERIA AGAINST INFECTION,
SO CONSIDER CRSPR IN BACTERIA
AND CGAS IS 1 OF THE INNATE
IMMUNE SYSTEM IN BACTERIA AND
INTERESTINGLY IN HUMANS WE NO
LONGER USE CRSPR ISSUE, OR
ENZYMES TO PROTECT US AGAINST
VIRUS INFEC, ABOUT YOU WE STILL
RELY ON CGAS TO PROTECT US
AGAINST VIRUS INFECTIONS SO THIS
IMMUNE DEFENSE AGAINST VIRUS
INFECTION FUNCTION OF CGAS HAS
BEEN CONSERVED THROUGH EVOLUTION
FROM BACTERIAL TO HUMAN, SO IT'S
HUMAN, THERE'S [INDISCERNIBLE]
CGAS, [INDISCERNIBLE] CGAS,
THERE'S SINGLE CELL EUKARYOTE,
THAT ALSO HAVE CGAS,
[INDISCERNIBLE] AS CGAS AND OF
COURSE BACTERIA THAT THERE ARE
MANY, MANY CGAS OR CGAS
ORIGINATE INDEED BACTERIAER RAW
DATA AND HAS BEEN CONSERVED FOR
BILLIONS OF YEARS.
OKAY SO I THINK I HAVE
ACKNOWLEDGED KEY PEOPLE WHO DID
THE WORK DURING MIRROR IMAGE
TALK AND MORE RECENT WORK ON
BACTERIAL CGAS WAS DONE BY
JUSTIN.
THANK YOU VERY MUCH.
[ APPLAUSE ]
NTHANK YOU ANYONE IS FREE TOO
USE THE MICs, 2 QUICK THINGS
ISSUES THE CME CODE WAS 5 WHERE
ARE 500 NIEF
FINE.
AND TO GET THE LIVE FEEDBACK
FORM, IT YOU HAVE TO REFRESH,
CAN YOU FIRST STARTED WATCHING
OR CAN YOU E-MAIL QUESTIONS
DIRECTLY.
NBEAUTIFUL WORK AND I HOPE I
DIDN'T MISS IT ON YOUR SLIDE,
THE LAST PART ABOUT THE CGAS
REGULATION, YOU ALSO HAD A
[INDISCERNIBLE] CGAS, I WANT TO
CALL IT DO YOU NEED THAT FOR THE
REDUCTION OR DO YOU NEED THE
REVERSIBLE CONJUGATION FOR WHAT
YOU SEE, DO YOU KNOW THAT
ALREADY?
NYEAH, THAT'S INTERESTING, SO
ABOUT [INDISCERNIBLE] ENZYME,
THIS IS THE CAP 3 PROTEIN, AND 1
WOULD EXPECT THAT, YOU KNOW THAT
THIS 1 IF YOU REMOVE THIS
UBIQUITIN CONYUIGATION, IT WOULD
HAVE AN OPPORTUNITY FOR FAT AND
IN SOME--IN SOME OF THESE
ACTUALLY HAD HAS A POSITIVE
EFFECT, IT ACTUALLY BEHAVES WHY
UOF THE LIKE CONJUSTICE ACCESS
GAGE, SO MAYBE IT REQUIRE A
CYCLE IN ORDER TO STIMULATE
ENZYME ACTIVITY, AND SO THAT'S
THE CAP 3, ENZYME IT'S VERY
INTERESTING BUT IT'S ALSO
ENIGMA.
YEAH.
>> THANK YOU.
THERE IS AT LEAST 1 ONLINE
BECAUSE WE'RE PAST TIME.
>> THE QUESTION IS-- HELLO?
>> YES,.
>> THE QUESTION IS MOST OF THE
NUCLEIC ARK SIDS ARE
INFLAMMATORY SO 1 WAY TO REDUCE
INFLAMMATION AND IS THE REASON
THE [INDISCERNIBLE] CHANGING
[INDISCERNIBLE] THAT ENDED UP
MAKING THE VACCINE THAT SAVED
LIVES ALL OVER THE WORLD NOW SO
DO WE HAVE A SIMILAR SYSTEM IN
THE MAMMALIAN SYSTEM WITH
MULTICELLULAR ORGANISM WHERE
THIS KIND OF REACTION MIGHT
HAPPEN BECAUSE WHENEVER YOU HAVE
A HIGH CELL DEATH AND ASCHEMIC
DEATH IN A STROKE OR HEART
ATTACK THAT IS A HUGE NUMBER OF
CELLS, HUGE NUMBER OF NEGLIG
ENSEL IS RELEASED, HOW DO THE
CELLS COMPARTMENTALIZE WITH THE
IN[INDISCERNIBLE] FRAGMENTS IN
THE MAMMALIAN SYSTEM.
>> RIGHT.
SO, YOU KNOW IT'S A VERY
IMPORTANT QUESTIONS AND SO I
TEND, MANY TIMES WE HAVE MANY
WAYS TO DEAL WITH DNA AND THE
CYTOPLASM, YOU DON'T WANT DNA TO
GET INTO THE CYTOPLASM SO CAN
YOU USE NUCLEACES TO DEGRADE DNA
AND THAT'S WHAT TRAX1 DOES.
AND THEN, YOU ALSO USE THIS
COMFORTMENTALLIZATION WITHIN THE
CELLS TO KEEP THE DNA IN THE
NUCLEUS OR IN THE MITOCHONDRIA
BUT IF THERE'S DAMAGE IN THE
MITOCHONDRIA, AND ALL OF HAVE
YOU DEFECTIVE AUTOPHAGY ISSUE BY
RICHARD YU, THEN THE ACTIVATED
PATHWAY CAN TRIGGER DECS SUCH AS
PARKINSON'S DISEASE, SIMILARLY,
[INDISCERNIBLE] IS COMPROMISED
SOMEHOW THAT CAN ALSO LEAD TO
ACTIVATION OF THESE PATHWAY,
GENOMIC INSTABILITY CAN LEAD TO
THE FORMATION OF MICROMUSEUM CLE
I, AND CGAS WOULD IMEAN TO THE
MICRONUCLEI AND GETS ACTIVATED
AND THAT CAN LEAD TO SENESCENSEL
AND SO, I THINK THAT WE USE THE
CGAS TO DETECTION VIRUSES ON
BACTERIA AND CERTAINLY THAT'S
IMPORTANT FOR HOST DEFENSE BUT
THIS PATHWAY ACTIVATION CAN HAVE
ADVERSE CONSEQUENCES AS SHOWN IN
MANY INFLAMMATORY DISEASES, A
LOT OF THESE DISEASES ARE CALLED
BY THE SO CALLED STERILE
INFLAMMATION AND WE THINK THAT
DNA IS AN IMPORTANT TRIGGER OF
THIS STERILE INFLAMM EGG EMPLOY
NIS THERE A MEASURE OFMENT CGAS
IN SOME OF THE AREAS OF ESCHEMIC
STROKE OR SOME OF THE OTHER
DISEASE IN LARGE ANIMALS?
ANY EVIDENCE OF CGAS RELEASE OR
ACTIVATION IN MAMMALIAN SYSTEMS?
ANY EVIDENCE?
>> YEAH, SO YEAH, CGAS
ACTIVATION AND YOU KNOW HUMAN
CELLS AND MOUSE MODELS AND MANY
DIFFERENT MODEL ORGANISMS.
YES, I'M NOT SURE IF I GET THE
QUESTION CORRECTLY BUT CERTAINLY
CGAS--CGAS ITSELF IS NOT
RELEASED OUTSIDE OF CELLS SO
IT'S ACTIVATED WITHIN A
CYTOPLASM BUT I SHOULD ALSO
POINT OUT IT'S ALSO CGAS IN THE
NUCLEUS, AND IN THE NUCLEUS,
CGAS IS BOUND TO HISTONES AND
THAT HISTONE BINDING PREVENTS
CGAS FROM GETTING ACTIVATED BY
GENOMIC DECKER NA. DNA.
>> THIS WAS BEAUTIFUL, IT'S
ALWAYS EXCITING FOR THE
BACTERIAL PHAGE STUFF IS REALLY
FASCINATING.
I HAVE 2 QUESTIONS, WOKNOW IS
THERE IS A REPORT--I MEAN
RECENTLY STING IS ACTUALLY
REPORTED TO ACTUALLY FORM A PORE
AS WELL, YOU DIDN'T MENTION THAT
BUT HOW MUCH DO YOU THINK IT
KRIEWBS, I THINK IT WAS CLAIMED
TO CONTROL AUTOPHAGE EXPE ALLOW
P3 ACTIVATION, DID YOU SEE THAT
AS WELL IN THE CONFIRMATIONAL
CHANGES HAVE YOU SEEN OR
ACTUALLY OPEN IT?
IT WOULD BE INTERESTING IN THOSE
PATIENTS THAT CONSTITTIVELY
ACTIVE STING WHAT WOULD BE THE
CORRELATE.
THE SECOND QUESTION IS MORE IN
REGARDS TO THE ACCOUNTERIAL
PHAGE MECHANISM WHICH IS
FASCINATING.
IS THERE A HUMAN CORRELATE
PARTICULARLY SENGS CGAS IS QUITE
ACTIVATED, IS THERE A SPONGE FOR
A HUMAN HOMOLOG OR OF THE V4
PROTEIN THAT HAVE YOU SEEN AND
IS ACTUALLY UBIQUITYALATION OF
CGAS A WAY IN WHICH HUMANS CAN
ACTIVATE AND MODIFY THAT PATHWAY
AS WELL.
>> OKAY, THANK YOU.
>> YEAH, OKAY, GREAT.
>> ALL RIGHT, FITTER FIRST
QUESTION IS BIOTAINED AND REASON
PAPER FROM [INDISCERNIBLE] LAB
AT MIT SHOWING THAT STING CAN
FORM PROTEOME CHANNEL AND I KNOW
IT'S A NICE PAPER, YOU KNOW AND
THE STRUCTURE OF STING WE DID
SEE A PAW AND WE DON'T KNOW WHAT
IT DOES AND WE ALSO KNOW THAT
WITH STING TRAFFICKING FROM
[INDISCERNIBLE], IT WOULD LEAD
TO WHAT WE CALL THE NONCAN
NONEICAL FORM OF AUTOPHAGY IT
WOULD LEAD TO LC3 DEGRADATION IS
IT IS CAUSE BIDE ENDOSTUDIES OF
MULTIPLE ENDOCRINAL MEMBRANE
DAMAGE OR LYSOSOMAL MEMORY
RESPONSE BREAN DAMAGE.
AND SO, I THINK THAT STAIN CAN
FORM A POL OR CHANNEL AND IT CAN
LEAK PROTON, AND THAT COULD BE A
MECHANISM TO ACTIVATE DATP S
THAT LEADS TO AUTOPHAGE
COMPETENT INDUCTION CAN THAT'S
AUTOSOMAL DAMAGE THAT COULD
ACTIVATE NRP 3 AND MACROPHAGES
BUT NOT IN OTHER CELLS.
NTHAT WOULD BE CONSTITTATIVELY
ACTIVATED IN THE PATIENT WHO IS
HAVE MUTATIONS RIGHT?
>> RIGHT.
SO I THINK IN TERMS OF THE POL
FORMATION, THEN THE QUESTION,
THIS DEPENDS ON THE EXTENT OF
STAIN ACCOUNTIVATION, I THINK IF
YOU ACTIVATE STING VERY
STRONGLY, THEN THEY CAN LEAD TO
CELL DEATH, BUT IF IT'S
WEAKACIVATION AND SOME OF THOSE
POLs CAN BE REPAIRED BY
[INDISCERNIBLE] CONTACTS SO I
DON'T KNOW WITH THOSE PATES IF
THOSE POLs ARE REPAIRED BY
[INDISCERNIBLE] COMPLEX OR THEY
LEADS TO CELL DEATH, IT'S NOT
CLEAR.
AND SO, YEAH, I THINK THAT IN
MACROPHAGES, THE FORMATION OF
THESE POLs CAN LEAD TO NFCAP B
FORMATION AND THAT CAN ALSO
TRIGGER THIS NONCANONICLE FORM
OF AUTOPHAGY.
THE SECOND QUESTION IS WHETHER
IN MAMMALIAN CELLS, CGAS USE A
URK CIBOL WITTEN LIKE MECHANISM
TO GET ACTIVATED AND THE ANSWER
I THINK IS PROBABLY NOT BECAUSE
THE TERMINAL [INDISCERNIBLE] IS
NOT CONSERVED IN HUMAN CGAS.
AND AND SO WE THINK, YOU KNOW IN
BACTERIA, I MENTION THEY ARE
LIKE 6000 BACTERIA LIKE CGAS
ENZYMES, THE CGAS, SOMEHOW GETS
INTO EUKARYOTIC CELLS, THROUGH
THESE SYMBIOSIS, YOU KNOW THEY
MAY NOT USE THIS MECHANISM, SO,
SO IT WILL BE A DIFFERENT
MECHANISM, SO I THINK THAT IN
BACTERIAL BECAUSE
THEY'RE--THERE'S SUCH A
DIVERSITY OF THIS CGAS LIKE
ENZYMES, THE MECHANISM OF
ACTIVATION COULD BE QUITE
DIFFERENT BETWEEN DIFFERENT
BACTERIAL SPECIES AND SO THE
SHORT ANSWER IS THAT FOR HUMAN
CGAS, THE ACTIVATION BASKLY IS
VERY SIMPLE, BY BINDING TO DNA,
THAT'S A MEASURE MECHANISM.
THERE MAY BE SOME
[INDISCERNIBLE] MECHANISM BUT
THE MEASURE MECHANISM IS DNA
BINDING AND THEN THE QUESTION
THE OTHER QUESTION IS WHERE DO
THE MAMMALIAN CELLS SPONGE UP
THESE CGAS?
SO SOME VIRAL PROTEINS THEY WILL
COMPETE WITH CGAS COMBINE TO DNA
AND SO FOR EXAMPLE IN K, SV,
THERE'S A VIRAL PROTEIN THAT CAN
BIND TO DNA AND MUCH MOREENTIOUS
FICIENTLY THAN CGAS AND IT
INHIBITS CGAS ACTIVATION,
THERE'S A P.O. BOX VIRUS HA CAN
ENCODE A ENZYME THAT, CAN
DEGRADE THESE 2 PRIME 3 PRIME
CGAS, SO THERE'S A POX VIRUS
PATHWAY AND THERE ARE OTHER
PROTEINS THAT COMBINE TO STING
AND INHIBIT THIS PATHWAY.
SO THE SHORT ANSWER IS THAT,
ANIMAL VIRUSES THAT USE A
VARIETY OF MECHANISMS TO INHIBIT
THE CGAS PATHWAY.
>> GREAT TALK YAIMS JAMES, HOWU
SEE THE INFLAMMATORY MODELS, IN
MOST CELLS IT'S ACTIVATED
ALONGSIDE CGAS ARE THERE CERTAIN
CIRCUMSTANCES IN WHICH THE
RESPONSE IS MORE IMPORTANT?
>> RIGHT, SO WITH M2 IT'S A VERY
INTERESTING QUESTION.
SO YOU KNOW I SHOW THAT IN
TRUCKS 1 DEFICIENT CELLS AND
MICE, IF YOU KNOCK OUT CGAS, YOU
COMPLETELY RESCUE THE PHENOTYPE
AND WE ALSO HAVE ANOTHER MODEL
THAT'S DNAS2 KNOCKOUT MICE,
THOSE MICE THEY HAVE EVEN MOSTLY
BEEN PHENOTYPE IS EMBRYONIC
LETHAL AND THAT CAN ALSO BE
RESCUE BY KNOCKING OUT CGAS OR
STING.
AND SO THAT IF YOU THINK ABOUT
IT, YOU MAY NOT PREDICT SUCH A
RESULT BECAUSE IN DNS 2 KNOCK
OUT CELLS YOUR CLERLY IN THE DNA
OF THE CYTOPLASM AND IF YOU TAKE
AWAY CGAS, IF THOSE CAN ACTIVATE
INFLAMMA STUDIES OF MULTIPLE
ENDOCRINE, YOU WILL HAVE TO HAVE
DISEASE, THE RESULT IS THAT ALL
WE NEED TO DO IS TO TAKE OUR
CGAS OR STING AND THEN WE CAN
RESCUE THE PHENOTYPE AND THAT
SUGGESTS THAT THOSE DNA IT THEY
GET INTO THE CYTOPLASM, THEY
DON'T ACTIVATE M2, THERE'S 1
POSSIBILITY OR THEY DON'T
ACTIVATE TLR 9 AND THIS 1
POSSIBILITY, NOT A POSSIBILITY,
SOMEHOW IN THOSE CELLS THE DMA
THEREYA ANOTHER MECHANISM TO GET
RID OF THE DNA.
NSO I COMPLETELY DIFFERENT
QUESTION, IN YOUR ORIGINAL
SCREEN WITH THE MASS SPEC, YOU
FOUND 3 PROTEINS, ARE THE OTHER
2 RELATED TO CGASES IN ANY WAY.
>> YEAH, YEAH.
--WE HAVE NOT GONE BACK IN WITH
THE PROTEINS.
INCOME OKAY, I'M SORRY--THERE
WAS ANOTHER QUESTION BUT I HAVE
TO CUT IT OFF NOW.
WE HAVE ANOTHER LECTURE COMING
IN.
BUT THERE WILL BE A RECEPTION,
SO PERHAPS PEOPLE CAN TALK TO
YOU A LITTLE MORE DURING THE
RECEPTION AND MAYBE I CAN
FORWARD A LOT OF QUESTIONS FROM
ONLINE, MAYBE I CAN FORWARD THEM
TO YOU.
NOKAY, APOLOGIZE TO THE PEOPLE
ONLINE, ANY CLOSING REMARKS
DOCTOR?
ALL RIGHT, THANK YOU VERY
MUCH.
THANK YOU VERY MUCH.
[ APPLAUSE ]