I'M I AM HEADING THE SECTION OF
THE MOLECULAR TRANSACTION AT
NICHD.
IT IS MY PRIVILEGE TO WELCOME
TODAY'S SPEAKER, DR. TOBIAS
WALTHER.
DR. WALTHER IS AN HHMI
INVESTIGATOR, NEWLY APPOINTED
CHAIR, PROGRAM CHAIR, OF THE
CELL BIOLOGY DEPARTMENT AT
SLOAN-KETTERING, PROFESSOR IN
BIOCHEMISTRY AT WEILL CORNELL
SCHOOL OF MEDICINE.
SOME OF YOU MIGHT REMEMBER AND
BE SURPRISED DR. WALTHER IS NOT
AT HARVARD.
HE RECENTLY MOVED FROM HARVARD
WHERE HE WAS PROFESSOR AT THE
CHAN SCHOOL OF PUBLIC HEALTH,
AND ASSOCIATE MEMBER OF THE
BROAD INSTITUTE.
THESE POSITIONS UNTIL LAST YEAR.
SO, THIS HAS BEEN A BUSY YEAR
FOR HIM BECAUSE HE MOVED TO
SLOAN-KETTERING, WHERE HE'S
RUNNING A LAB TOGETHER WITH DR.
ROBERT FORESER JR., FOR A LONGER
PERIOD OF TIME.
THEY INVESTIGATE CELL LIPID, IN
PARTICULAR MECHANISM OF NEUTRAL
FAT STORAGE.
MORE BROADLY, HIS LAB IS
INTERESTED IN HOW CELLS REGULATE
THE SUPPLY OF FAT, AND ABUNDANCE
OF LIPIDS, HOW THE CELLS STORE
AND MOBILIZE LIPIDS UPON NEED,
AND HOW PROCESSES HELP
PHYSIOLOGY AND MEMBRANE BIOLOGY
AND DYNAMICS.
OF COURSE, IT IS IMPORTANT THAT
ALL OF THESE FUNCTIONS ARE
RELATED TO HUMAN DISEASES, SO
THEY ARE VERY INVOLVED.
THEY USE NOTABLE APPROACHES,
SOME BIOPHYSICAL, SOME IMAGING,
BIOCHEMISTRY, PROTEOMICS,
LIPIDOMICS TO DISCOVER INSIGHTS
INTO LIPID METABOLISM.
HE IS A NATIVE OF GERMANY,
UNDERGRADUATE DEGREE IN
MOLECULAR GENETICS IN MUNICH,
GERMANY, AND DID HIS Ph.D.
STUDIES IN BIOCHEMISTRY AT THE
MOLECULAR BIOLOGY LAB IN
HEIDELBERG, DID A POSTDOCTORAL
STUDY IN THE UCSF IN 2002-2006,
RETURNED TO GERMANY TO MAX
PLANCK INSTITUTE AT GROUP LEADER
FOR A SHORT PERIOD.
HE THEN MOVED BACK TO THE U.S.
IN 2010, AND BECAME AN ASSOCIATE
PROFESSOR IN CELL BIOLOGY AT
YALE.
FROM THERE, IN 2014, HE JOINED
HARVARD AND THE BROAD INSTITUTE.
I THINK THIS IS JUST ENOUGH TO
LIST ALL OF THE MOVEMENTS, I'M
REALLY IMPRESSED BY THE AMOUNT
OF MOVEMENTS HE DID.
BUT IT SEEMS TO BE ALL GOING
UPWARD.
SO, ASIDE FROM RECOGNITION FROM
THE HHMI INSTITUTE, AND SEVERAL
NIH GRANTS AND AWARDS, I ONLY
HAVE TO INTRODUCE HIS TALK,
WHICH THE TITLE IS FAT MECHANISM
AND PHYSIOLOGY OF LIPID STORAGE.
PLEASE WELCOME DR. TOBIAS
WALTHER.
[APPLAUSE]
>> THANK YOU FOR THE
INTRODUCTION AND ALL OF YOU FOR
SHOWING UP FOR THE SEMINAR
TODAY.
IT'S A GREAT PLEASURE TO BE
HERE.
I THINK I HAVE HOPEFULLY THE
NEXT COUPLE MINUTES TO HOPEFULLY
IMPRESS YOU WITH ANYTHING ELSE
BUT MY ABILITY TO MOVE.
SO, BEFORE I GET STARTED, I
WANTED TO BRIEFLY MENTION
SOMETHING THAT TOMAS ALREADY
INTRODUCED, WHICH IS ALL THE
WORK THAT I'M GOING TO PRESENT
TODAY IS THE RESULT OF A
SCIENTIFIC PARTNERSHIP BETWEEN
MYSELF AND BOB FARESE, WORKED
TOGETHER FOR 15 TO 20 YEARS NOW,
WORKED AS A SCIENTIFIC
PARTNERSHIP.
WE RECENTLY TRIED TO REFLECT ON
OUR EXPERIENCES HERE A LITTLE
BIT ON HOW IT IS TO RUN A LAB
TOGETHER.
NOW, WHAT I WANTED TO INTRODUCE
TO YOU IS REALLY WONDERFUL, HOW
MUCH LIFE REQUIRES IN TERMS OF
ENERGY.
IT'S JUST IF YOU TAKE A REGULAR
HUMAN BEING, WE GENERATE ABOUT
OUR BODY WEIGHT IN ATP,
OBVIOUSLY TURNOVER, BUT ROUGHLY
BODY WEIGHT IN ATP EVERY DAY TO
DO ALL KINDS OF WONDERFUL
THINGS, MOVE AROUND, DIGEST,
THINK, WHAT NOT.
SO, A CLOSE IS BALANCE BY ENERGY
INTAKE, YOU HAVE TO FUEL THE
SYSTEM TO ALLOW THAT TO HAPPEN.
NOW, IN MOST SYSTEMS, I CAN'T
FIGURE OUT WHICH MICROPHONE,
SORRY.
IN MOST SYSTEMS, OF COURSE, THIS
BALANCE NEEDS TO HAPPEN, DOESN'T
PERFECTLY OCCUR OVER TIME BUT
THERE ARE FLUCTUATIONS BOTH IN
THE NEED TO EXPEND METABOLIC
ENERGY AND IN THE ABILITY TO
TAKE UP ENERGY FROM THE
ENVIRONMENT.
AND BECAUSE OF THOSE
FLUCTUATIONS THERE'S A NEED TO
BUFFER TEMPORARILY THE ABILITY
TO EXPAND ENERGY AND WE DO THAT
PREDOMINANTLY BY TEMPORARILY
STORING METABOLIC ENERGY.
AND BY AND LARGE THIS OCCURS IN
THE FORM OF LIPIDS.
THIS IS DRAWN ROUGHLY TO SCALE
FOR A HEALTHY HUMAN, AND SHOWS
ROUGHLY HOW MUCH METABOLIC
ENERGY WE TEMPORARILY STORE IN
THE FORM OF LIPIDS VERSUS
CARBOHYDRATES,' VOLVED AS A NEAR
UNIVERSAL FORM OF METABOLIC
ENERGY STORAGE BECAUSE MOLECULES
OF LIPIDS ARE STORED IN A
MOLECULE CHARACTERIZED BY
GLYCEROL BACKBONE WITH THREE
CHAINS.
THAT MAKES THAT MOLECULE
EXTREMELY HYDROPHOBIC, DOESN'T
MIX WITH WATER.
AS A CONSEQUENCE THIS IS THE
DENSEST FORM YOU CAN STORE
CARBOHYDRATES IN BECAUSE YOU
DON'T HAVE THE WATER.
THIS IS THE REASON PRESUMABLY
THIS HAS EVOLVED TO BECOME MORE
OR LESS UNIVERSAL FORM OF ENERGY
STORAGE.
IT CREATES A PROBLEM.
SO BECAUSE OUR CHEMISTRY HAPPENS
PREDOMINANTLY IN AQUEOUS SPACE,
WHEN YOU PUT TRIDELAYS TRIGLYCY
STAY SEPARATE.
YOU CAN RECONSTITUTE, IN A GLASS
ON MY PORCH.
OIL DOESN'T MIX WITH WATER, SO
HERE'S WHERE YOU WILL AT ENERGY
IS STORED IN A CELL, IF YOU
WILL.
THE WATER IS REALLY WHERE ALL
THE LIFE HAPPENS.
AT THE INTERFACE A LOT OF
BIOLOGY NEEDS TO HAPPEN TO ALLOW
THE POSITION OF THE ENERGY INTO
THE OIL PHASE, SOME OF YOU MAY
REMEMBER THAT IN OLD DAYS IN
ORDER TO EXTRACT DNA WE USED
SUCH INTERFACES TO DENATURE ALL
THE PROTEINS AND GET RID OF THEM
TO EXACT THE DNA IN THE WATER
SOLUBLE PHASE, CREATING UNIQUE
CHALLENGE.
IN CELLS THE OIL IS NOT STORED
AT MASSIVE LAYER OF FAT BUT
INSTEAD IN WHAT WE KNOW AS
ORGANELLES, LIPID DROPLETS, VERY
UNUSUAL COMPARED TO MITOCHONDRIA
OR RETICULUM, WITH A CORE OF
NEUTRAL LIPIDS THAT FORMS
ORGANIC PHASE, BOUNDED BY A
MONOLAYER, NOT A BILAYER
MEMBRANE, PHOSPHOLIPIDS INTO
WHICH SPECIFIC PROTEINS ARE
EMBEDDED.
AND REALLY THE DISCOVERY THAT
THERE ARE SPECIFIC PROTEINS TO
THIS SURFACE REALLY STARTED TO
PUT THIS ORGANELLE ON THE MAP AS
SOMETHING THAT'S NOT JUST AN
INERT BLOB OF FAT.
BUT AN ORGANELLE IN CELLS WITH
ALL THE USUAL DYNAMIC BEHAVIORS.
YOU CAN SEE LIPID DROPLETS
INTERACT WITH OR ORGANELLES SUCH
AS MITOCHONDRIA OR ENDOPLASMIC
RETICULUM.
IF YOU TAKE A SINGLE LIPID
DROPLET IF IT'S 300 TO 500
NANOMETERS CAN STORE EQUIVALENT
OF 23 BILLION ATP EQUIVALENTS,
THAT'S REALLY WHERE A LOT OF
METABOLIC ENERGY IS TEMPORARILY
STORED.
AS I MENTIONED IN CELLS YOU HAVE
MULTIPLE OF THESE LIPID
DROPLETS, MOST OFTEN.
AND THEIR PRESENCE CONVERTS TO
EMULSION, DROPLETS FORM, AND
SURFACTANTS ARE THERE TO
BASICALLY DELINEATE TWO SURFACES
AND ALLOW ALL THE TRAFFIC IN AND
OUT, ALL THE BIOLOGY THAT I'M
GOING TO TALK ABOUT.
NOW, IN MULTI-CELLULAR
ORGANISMS, FAT IS STORED?
ADIPOSE TISSUE.
YOU CAN SEE THIS IS A SINGLE
LIPID DROPLET THAT OCCUPIES MOST
OF THE ADIPOCYTE ORGANELLES ARE
SQUEEZED.
LIPID DROPLETS ARE NOT
RESTRICTED TO ADIPOCYTES, THEY
OCCUR IN PRETTY MUCH EVERY OTHER
CELL TYPE PEOPLE HAVE LOOKED AT
INCLUDING WHERE YOU SEE AFTER
FATTY ACIDS COME FROM THE MEAL,
AND THE LIVER WHERE THEY ARE
TRAFFICKED FROM EITHER THE
ADIPOSE TISSUE, STORED IN LIPID
DROPLETS.
THE HEART RUNS ON FATTY ACIDS,
METABOLIC FUEL, TEMPORARILY CAN
STORE LIPID DROPLETS.
FOR INSTANCE MILK HAS A LOT OF
FAT, THOSE ARE LIPID DROPLETS
THAT GET SHED FROM THE
EPITHELIAL CELL, STILL POORLY
UNDERSTOOD MECHANISM.
LIPID DROPLETS ARE PRESENT
THROUGHOUT PHYSIOLOGY, NOT ONLY
IN PHYSIOLOGY BUT
PATHOPHYSIOLOGY.
SO, FOR INSTANCE, MANY OF YOU
KNOW OF COURSE THAT WE'RE FACING
AN UNPARALLELED EPIDEMIC OF
OBESITY IN THIS COUNTRY AND
ELSEWHERE WHERE REALLY MASSIVE
ACCUMULATION OF TRIGLYCERIDES
INTERFERE WITH PHYSIOLOGY OF
HUMANS.
WHAT'S LESS KNOWN THERE'S ALSO A
DIFFERENT CONDITION KNOWN AS
LIPODYSTROPHY, FAILURE TO
MAINTAIN ADIPOSE TISSUE LEADS TO
SEVERE DISEASE.
IN BOTH CASES ONE CAN THINK FOR
DIFFERENT REASON ADIPOSE TISSUE
GETS OVERWHELMED WITH FATTY
ACIDS, ALTHOUGH THIS PERSON HERE
LOOKS PHENOTYPICALLY HEALTHY THE
TWO CONDITIONS SHARE SIMILAR
HALLMARKS IN TERMS OF DISEASE.
ON THE BIOCHEMICAL LEVEL,
TRIGLYCERIDES ARE SYNTHESIZED BY
TWO ENZYMES, KNOWN INITIALLY
DISCOVERED BY EUGENE KENNEDY,
CO-WORKERS IN THE 19 50s,
PURIFIED AND NAMED AFTER THEIR
ACTIVITIES, KNOWN AS D GAT 1 AND
2.
AND THOSE TWO TAKE A POLAR
MOLECULE, AND MAKE IT APOLAR.
AFTER BOB DISCOVERED DGAT 1,
THIS IS A FANTASTIC DRUG TARGET,
IF YOU'RE A MOUSE.
IF YOU DELETE OR INHIBIT MICE
LIVE 30% LONGER, RESISTANT TO
DIET-INDUCED OBESITY, AND THEY
HAVE ESSENTIALLY ALL POSITIVE
CHARACTERISTICS OF METABOLISM.
SO THIS HAS INSPIRED A LOT OF
PHARMACEUTICAL COMPANIES TO
DEVELOP INHIBITORS.
I'LL SAY A LITTLE BIT ABOUT THAT
IN A SECOND.
TURNS OUT THAT WHEN YOU THINK
ABOUT TRIGLYCERIDE SYNTHESIS TWO
PARTS OF THE EQUATION ARE WORTH
CONSIDERING.
ONE IS THEY MAKE AND ARE PUT IN
DROPLETS BUT REMOVE DIGLYCEROL,
TWO SIDES OF THE COIN HERE.
WHEN YOU FOR INSTANCE GENERATE A
MOUSE THAT DOES NOT HAVE BOTH
ENZYMES SPECIFICALLY IN ADIPOSE
TISSUE, THIS MOUSE IS
SURPRISINGLY HEALTHY, DOESN'T
HAVE ANY PARTICULAR PROBLEM, YOU
CAN SEE IT HAS ADIPOSE TISSUE,
BUT THE ADIPOSE TISSUE DOESN'T
HAVE ANY FAT.
IT'S JUST A SIMPLE WAY TO
VISUALIZE THE EFFECT.
THE MICE ARE FINE AS LONG AS
THEY HAVE FOOD, BUT IF YOU DON'T
HAVE FOOD AND PUT THEM IN THE
COLD, A MAJOR METABOLIC STRESSOR
IN MICE, THEY CAN NOT MAINTAIN
BODY TEMPERATURE.
SO THIS WILL ARGUE AS YOU
PROBABLY KNOW, WHAT I ARGUED FOR
IS INTEREST TRIGLYCERIDES
FUNCTION AS ENERGY SOURCE.
WHAT ALSO HAPPENS IS THAT IN
MANY CASES SUCH AS OBESITY THIS
EXCESS LIPIDS OVERWHELM
MACHINERY AND FLOW INTO OTHER
METABOLITES THAT WHEN ACCUMULATE
CAN TRIGGER E.R. STRESS THAT
LEADS TO CELL DEATH OR
ADAPTATION.
TURNS OUT IN HUMANS THAT'S WHAT
HAPPENS IN THE INTESTINE BECAUSE
THOSE MICE I MENTIONED EXPRESS
DGAT1 AND 2, WE ONLY EXPRESS
DGAT1.
WHEN WE DISCOVERED PATIENTS WITH
MUTATIONS IN THIS ENZYME, THEY
HAVE A SEVERE CONGENITAL
DIARRHEA SYNDROME, ESSENTIALLY
WHAT WE THINK IS GOING ON IS
THAT WHEN THE ORGANISM IS
CONFRONTED WITH A LOT OF FATTY
ACIDS FROM THE MEAL ENTEROCYTE
CANNOT TEMPORARILY BUFFER THE
INFLUX IN LIPID DROPLETS, AS A
RESULT TRIGGERS METABOLIC --
DYSFUNCTION AND STRESS,
CONSISTENT WITH THIS WE NOW CAN
FIGURE OUT HOW TO -- SORRY, HOW
TO DEAL WITH THIS, WITH
BYPASSING THE INTESTINE FOR
THOSE PATIENTS.
MORE BROADLY, THIS HAS LED TO A
CONCEPT PROPOSED BY US AND
OTHERS THAT LIPID DROPLETS HAVE
A PROTECTIVE FUNCTION IN MANY
CONTEXTS SUCH AS LIVER
MACROPHAGES FOR MUSCLE AND
HEART, WHERE THEY OFTEN PROTECT
THE TISSUE FROM THE ACCUMULATION
OF METABOLIC DISEASE.
AND WE THINK THIS IS ALSO WHY
DGAT1 INHIBITORS, KILLS THE
PROTECTIVE FUNCTION.
WE KNOW FROM WORK OF MANY
LABORATORIES INCLUDING OURS THAT
THE PROCESSES INVOLVED IN LIPID
STORAGE ARE INTIMATELY LINKED TO
INHERITED METABOLIC DISEASES
BECAUSE WHEN YOU HAVE MUTATIONS
IN ENZYMES INVOLVED IN THE
SYNTHESIS OF TRIGLYCERIDE YOU
GET A NUMBER OF SEVERE DISEASES.
NOW, WITH THAT, YOU COULD ASK IS
ALL HOPE LOST?
TURNS OUT THERE'S ALSO
INHIBITORS FOR THIS OTHER
ENZYME, THERE'S A LESSON HERE
BECAUSE WHEN WE INITIALLY FOUND
THAT ENZYME AND MADE A DELETION
IT WAS LETHAL IN MICE.
MICE DIE SOON AFTER BIRTH.
TURNS OUT THAT NOW THIS HAS
BECOME A FRONTRUNNER FOR
TREATMENT OF FATTY LIVER
DISEASE, AGAIN BECAUSE OF THE
SPECIES DIFFERENCE.
AND WE FIND THAT IF YOU JUST
RESTRICT THE EFFECT TO MICE,
SORRY, TO LIVER, THAT PROTECTS
THE ANIMALS FROM FATTY LIVER
DISEASE.
ONE OF THE MAJOR SIDE EFFECTS OF
OBESITY BECAUSE NOW FATTY ACIDS
ARE NOT ONLY IN THE ADIPOSE
TISSUE ANYMORE BUT SPILL OVER TO
THE LIVER.
THIS PROTECTS FROM THAT AND HAS
LED TO MOVING THOSE DRUGS INTO
CLINICAL TRIALS WITH MARKEDLY
REDUCED LIVER FAT.
THIS IS INITIALLY DONE BY
PFIZER.
TURNS OUT WHAT I TOLD YOU
SHOULDN'T MAKE SENSE, RIGHT?
YOU STILL HAVE THE PROTECTIVE
EFFECT, WHY IS ANY NOT A PROBLEM
WITH DGAT2?
YOU NOT ONLY BLOCK TRIGLYCERIDE
SYNTHESIS BUT A FEEDBACK
REGULATION SHUTS OFF SYNTHESIS
OF MORE FATTY ACIDS, PREVENTS
ACCUMULATION OF ANY LIPID TOXIC
INTERMEDIATE.
ALL OF WHAT I TOLD YOU IN A
RELATIVELY SHORT PROGRESSION IS
JUST MEANT TO GIVE AN
INTRODUCTION THAT LIPID DROPLETS
CAN HAVE TWO MAJOR FUNCTIONS IN
CELLS.
THEY CAN BE USED TO STORE
METABOLIC EMERGENCY AND DETOXIFY
INTERMEDIATES OF METABOLISM.
THREE TOPICS IN THE BASIC
BIOLOGY OF ORGANELLES, HOW THEY
ARE MADE, WHAT ARE ENZYMATIC
MECHANISMS, HOW DO YOU PACKAGE
THEM INTO LIPID DROPLETS AND HOW
ARE PROTEINS THAT MEDIATE
EXCHANGE WITH THE REST OF THE
CELL ACTUALLY GETTING THERE, AND
FINALLY HOW DO YOU EXTRACT AND
MOBILIZE THINGS FROM A LIPID
DROPLET AGAIN.
FIRST PART, HOW ARE CELLS
SYNTHESIZED TRIGLYCERIDES,
THERE'S TWO ENZYMES BUT THEY
ONLY SHARE A NAME.
OTHERWISE THOSE ARE
EVOLUTIONARIAL UNRELATED, DGAT 1
IS A MEMBRANE PROTEIN, 2 AS A
HAIRPIN DOMAIN, OTHERWISE
CYTOSOLIC.
WE SOLVED THE STRUCTURE BY
MICROSCOPY, REVEAL AS DIMER
HERE, IF YOU FOCUS ON ONE SIDE
WHAT WE FOUND IS THE CATALYTIC
CENTER IS DEEPLY BURIED WITHIN
THE PLANE OF THE E.R. MEMBRANE.
ACCESSIBLE FROM BOTH THE
CYTOSOLIC SIDE THROUGH A TUNNEL
AND ALSO THROUGH A LATERAL GATE
WITHIN THE MEMBRANE.
AND BASED ON MANY EXPERIMENTS WE
NOW BELIEVE THIS CATALYTIC
CENTER IS ACCESSED FROM THE SITE
OF THE CYTOPLASM, LOOKING LIKE
THIS CARROT HERE, ACCESSES
CATALYTIC SITE, A MOIETY
ACCESSES CATALYTIC CENTER, THE
MOLECULE IS FORMED THAT CAN BE
RELEASED INTO THE PLANE OF THE
MEMBRANE.
INTRIGUINGLY, JUST AS A SIDE
NOTE, THIS ENZYME IS A MEN OF A
FAMILY MEMBRANE BOUND
TRANSFERASES, A LARGE GROUP THAT
ARE INVOLVED IN THE FORMATION OF
IMPORTANT SIGNALING MOLECULES
SUCH AS HEDGEHOG OR WINGLESS.
WE NOW HAVE STRUCTURES FOR A
NUMBER BUT I'LL SKIP THIS.
WE HAVE MUCH LESS INFORMATION ON
DGAT2, BUT IT WAS ONE OF THE
MAJOR PROTEINS HIGHLIGHTED IN
THE INITIAL ALPHA FOLD
PREDICTION, TO SHOW THIS IS HOW
WE THINK THIS ENZYME IS LOOKING
LIKE BASED ON THE PREDICTION,
AND INTRIGUINGLY HAS AN ACTIVE
SITE, NOT IN THE MEMBRANE BUT ON
THE CYTOSOLIC SIDE OF THE
MEMBRANE.
SO NOW THAT WE UNDERSTAND THAT
PARTICULARLY THIS ENZYME
RELEASES THE TRIGLYCERIDE, THE
NEXT QUESTION BECOMES HOW THEN
DO YOU FORM A LIPID DROPLET?
AFTER THE TRIGLYCERIDE IS
RELEASED INTO THE MEMBRANE?
FOR MANY YEARS THE GENERAL
THINKING WAS, WELL, YOU JUST
RELEASE IT INTO THE MEMBRANE,
AND WE KNOW FROM WORK OF
HAMILTON AND OTHERS THAT THERE'S
A MAXIMUM SOLUBILITY, AROUND 3.
UP TO 3% OF TRIGLYCERIDE, THEY
JUST SOLUBILIZE IN THE MEMBRANE,
AND IF YOU THINK ABOUT IT, VERY
SIMILAR TO SIDE CHAINS OF A
PHOSPHOLIPID.
ABOUT 3% PHASE SEPARATION OCCURS
AND AN OIL PHASE STARTS
EMERGING.
FOR MANY YEARS IT WAS THOUGHT
THAT THAT'S JUST A PASSIVE
PROCESS THAT OCCURS.
BUT WE AND OTHERS HAVE FOUND
THAT ACTUALLY CELLS DON'T LEAVE
THIS PROCESS UP TO CHANCE BUT
INSTEAD HAVE EVOLVED DEDICATED
PROTEIN MACHINERY THAT ORGANIZED
FORMATION OF LIPID DROPLETS OUT
OF THE E.R.
SPECIFICALLY, A KEY PROTEIN FOR
THIS IS A PROTEIN CALLED SAPIN,
NAMED AFTER ONE OF THE
DISCOVERERS OF A MUTATION IN THE
'50s THAT LEADS TO THIS LIPID
DYSTROPHY.
SO, THIS PROTEIN IS CALLED SAPIN
OR BSCL 2, AND MUTATIONS
ESSENTIALLY LEAD TO A COMPLETE
LACK OF ADIPOSE TISSUE, AND THIS
IS THOUGHT TO OCCUR BECAUSE
DURING THE DIFFERENTIATION OF
ADIPOSE TISSUE MASSIVE SYNTHESIS
OF TRIGLYCERIDES OCCURS, WHEN
THE MACHINERY THAT I WILL SHOW
YOU INVOLVED IN TRIGLYCERIDE
PACKAGING INTO LIPID DROPLETS IS
DEFICIENT YOU GET E.R. STRESS,
TOXICITY, PRESUMABLY THAT'S WHY
THOSE CELLS DON'T SURVIVE.
FIFTY YEARS LATER THE GENE
ENCODING THIS PROTEIN WAS
CLONED, AND IT WAS SHOWN BY A
NUMBER OF LABS THAT THE
DEFICIENCY IN THIS PERTURBED
LIPID DROPLETS IN YEAST, PLANTS,
WORMS, MANY DIFFERENT LABS,
INCLUDING OUR OWN, WHERE WE
FOUND INITIALLY IN IN MAMMALIAN
CELLS GENE ENCODING THIS, THIS
IS A REPRESENTATION LOOKING FOR
PHENOTYPES ASSOCIATED WITH
DEPLETION OF EACH GENE GIVES ONE
OF THE STRONGEST PHENOTYPES ON
LISPED DROPLETS.
SO THAT PHENOTYPE CAN BE
SUMMARIZED IN SHORT, IN A FLY
CELL HERE, THAT IN NORMAL CELLS
THERE'S A FEW EXISTING
PREEXISTING LIPID DROPLETS,
SHOWN IN THE GREEN COLOR,
SURROUNDED BY LIPID DROPLET,
LIVEDROP.
THERE'S PREEXISTING DROPLETS,
WHEN YOU ADD FATTY ACIDS,
EQUIVALENT OF McDONALD'S,
THERE'S A FEW LIPID DROPLETS
FORMING IN AN ORGANIZED WAY AT
SOME SITES IN THE LIPID DROPLET,
IN THE E.R.
WHEN YOU DO NOT HAVE SEIPIN
PREEXISTING DROPLETS, CAN YOU
SEE SMALL BLISTERS OCCURRING,
OBSERVED ACROSS DIFFERENT
SPECIES, THAT PHENOTYPE.
WE TACKED IT AT ENDOGENOUS LOCUS
WITH FLUOROPHORE, TRACKED.
YOU CAN SEE AN E.R. SIGNAL IN
RED.
AND SEIPIN IN GREEN.
WHAT BECOMES OBVIOUS IS THAT THE
PROTEIN FORMS FOCI, COMPLEXES IN
THE E.R. MEMBRANE.
AGAIN, YOU CAN SEE THIS EASY IN
A DROSOPHILA CELL THAT IS THE
SAME AS TRUE IN MAMMALIAN CELL,
IN THIS CASE A HUMAN CELL.
THAT OF COURSE IMMEDIATELY
BROUGHT US TO WHAT ARE THESE
COMPLEXES, HOW DO THEY WORK,
WHAT DO YOU HAVE TO DO AND WHY
DO YOU GET THAT PHENOTYPE?
SO IN ORDER TO UNDERSTAND THAT
WE SOLVED THE STRUCTURE OF SUCH
A COMPLEX.
THIS IS BY CRYO-ELECTRON
MICROSCOPY.
WHAT BECOMES OBVIOUS, THIS FORMS
A LARGE OLIGOMERIC RING
STRUCTURE IN THE ENDOPLASMIC
RETICULUM MEMBRANE WITH A
LUMINAL DOMAIN, AND MANY
TRANSMEMBRANE SEGMENTS.
SO TO GIVE A BETTER VIEW WHAT
YOU CAN SEE IS THERE'S A CASE
STRUCTURE, AGAIN OPEN TO THE
PLANE OF THE MEMBRANE AND
RELATIVELY CLOSE.
WHAT WE THINK HAPPENS HERE IS
THAT THIS PROVIDES A SPACE IN
THE CENTER OF THIS, NOT UNLIKE
FOR INSTANCE GRUEL E L DURING
PROTEIN FOLDING, TRIGLYCERIDE
MOLECULES CAN DIFFUSE IN AND
FIND A SPACE THAT EXCLUDES
PHOSPHOLIPIDS AND THERE BY HELPS
CATALYZING PHASE TRANSITION, IN
OTHER WORDS THE LIKELIHOOD OF
THREE TRIGLYCERIDE MOLECULES TO
MEET RATHER THAN INTERACT WITH
PHOSPHOLIPID.
WE TESTED THIS IN MOLECULAR
DYNAMIC SIMULATIONS, FINDING FOR
INSTANCE TOGETHER WITH
UNIVERSITY OF CHICAGO WE FIND
ONLY WHEN SEIPIN IS PRESENT,
LIPID DROPLET FORMATION OCCURS
IN THE CENTER OF THE VERY LARGE
COMPLEX.
AND THEN YOU CAN SEE IN THE
DYNAMIC SIMULATIONS THAT THE
COMPLEX OPENS AND LIPID DROPLET
FORMS, TO THE CYTOPLASM.
WE CAN SEE, IMPORTANTLY, ONLY
WHEN WE HAVE SEIPIN CAN YOU
START NUCLEATING LIPID DROPLET
OR PHASE TRANSITION OF
CONCENTRATIONS BELOW THAT
SOLUBILITY LEVEL.
THAT APPEARS TO BE A CRUCIAL
PART OF THE MOLECULE AT LEAST IN
SIMULATIONS TO HELP WITH
NUCLEATION.
WHEN YOU LOOK MORE CAREFUL AT A
STRUCTURE IT HAS A RING OF
LUMINAL DOMAINS, PROTRUDING IN
THE MEMBRANE, WE THINK THIS
HELPS EXCLUDE PHOSPHOLIPIDS, AND
THESE ARE VERY IMPORTANT, ALSO
SHOWN BY OTHERS FOR NUCLEATION
AND TRANSMEMBRANE DOMAINS.
WHAT WE DIDN'T REALIZE, THESE
HELIXES THAT PUSH APART
PHOSPHOLIPIDS ARE NOT ONLY
CONSERVED IN THEIR CHARACTER BUT
ALSO IN EVOLUTION ARE CONSERVED
WITH THEIR -- IN THEIR SPECIFIC
SEQUENCE.
THAT'S SOMEWHAT UNUSUAL.
WHY IS THERE ANY SPECIFIC
SEQUENCES CONSERVED?
SO THE LAB POSTULATED WE MAY BE
MISSING A CO-FACTOR AND SHE DID
SOME EXPERIMENTS, I'LL SHOW ONE
HERE.
SHE EITHER HAD THESE HYDROPHOBIC
HELICES, FOUND ONE PROTEIN
SPECIFICALLY INTERACTED WITH
THIS FORM OF THE COMPLEX BUT NOT
THAT ONE, UNCHARACTERIZED, A
MEMBRANE PROTEIN WE HAVE RENAMED
TMEM FROM TMEM 159.
WHEN WE TAKE CELLS, TACK SEIPIN
ENDOGENOUS LOCUS AND NEW PROTEIN
LDAF, AND WE FOLLOW LIPID
DROPLET FORMATION BY A THIRD
COLOR KNOCK-IN, AND USING A
MARKER PROTEIN THAT LIPID
DROPLETS ONLY FORM AT SITES THAT
HAVE BOTH SEIPIN AND THIS LDAF
PROTEIN.
AND YOU CAN -- YES, THAT'S WHY
WE RENAMED THE TWO PROTEINS
TOGETHER, FORMED THE LIPID
DROPLET ASSEMBLY COMPLEX.
ONE OF THE THINGS THAT WAS
GRATIFYING, IS SEIPIN WOULD BE
SUFFICIENT TO MEDIATE, WHEN WE
PURIFIED SEIPIN BIOCHEMICALLY
NEVER FOUND ASSOCIATE TO
TRIGLYCERIDE, ONLY WHEN YOU
CO-PURIFY AND RECONSTITUTE YOU
CAN SEE IT'S TRIGLYCERIDE
BECAUSE IT IS SENSITIVE TO THE
INHIBITION AND CAN BE RAISED AT
EXTRA FATTY ACIDS.
THIS APPEARS TO START SORT OF
TRANSFORMING THE MICELLE.
IF YOU LOOK AT SEIPIN IT FORMS A
FLAT MICELLE.
WE SEE THERE'S TWO THINGS IN
HERE, ADDITIONAL DENSITY OF
PROTEIN, VERY POORLY RESOLVED,
WE CAN TALK BIT, AND
TRIGLYCERIDE.
WE THINK THIS IS THE EARLIEST
INTERMEDIATE OF LIPID DROPLET
FORMATION WE CAN VISUALIZE WHERE
WE START HAVING THIS COMPLEX AND
SOME TRIGLYCERIDE TRAPPED IN THE
MIDDLE.
FASCINATINGLY AS LIPID DROPLETS
FORM THE COMPLEX DISASSOCIATES,
SEIPIN STAYS IN THE E.R.
MEMBRANE, AND LF1 STARTS TO
DECORATE THE SURFACE OF LIPID
DROPLETS.
SO IF I PUT THIS TOGETHER INTO A
MODEL, WHAT I TOLD YOU ABOUT, IT
WILL LOOK LIKE THIS.
YOU HAVE ENZYMES THAT TAKE THE
FATTY ACID, RELEASED INTO THE
PLANE OF THE MEMBRANE, DIFFUSE
INTO THE CAGE SHAPE OF THE
SEIPIN LDAF COMPLEX, THAT ALLOWS
PHASE TRANSITION.
AND THEN ONCE YOU START THAT,
THE COMPLEX OPENS AND GOES
TOWARDS THE CYTOPLASM.
AT THAT POINT ADDITIONAL
MONOMERS OF TRIGLYCERIDES ARE
INTEGRATED INTO THIS BY A
PROCESS EQUIVALENT THAT'S EASIER
TO GET INTO THE PHASE THAN TO
GET OUT.
THAT'S OUR CURRENT UNDERSTANDING
OF HOW LIPID DROPLET WORKS.
THERE'S A LOT OF MYSTERY STILL
HERE.
NOW, ONCE IT FORMS TO INTERACT
WITH THE REST OF THE CELL IT IS
REALLY IMPORTANT TO HAVE THE
SPECIFIC SET OF MEMBRANE
PROTEINS ON THE SURFACE.
AND A KEY QUESTION IS HOW ARE
PROTEINS TARGETED TO THE
SURFACE?
WE STILL DON'T UNDERSTAND THAT
COMPLETELY.
WE BELIEVE THIS IS A FUNDAMENTAL
QUESTION IN BIOLOGY BECAUSE IT'S
NOT SIMILAR TO OTHER THINGS.
THERE'S NO IMPORT MACHINERY
KNOWN FOR LIPID DROPLETS SUCH AS
FOR E.R., MITOCHONDRIA.
THERE ARE NO SPECIFIC LIPIDS
THAT MARK THE SURFACE SUCH AS
ENDOSOME OR PLASMA MEMBRANE.
BUT INSTEAD THE STRUCTURE OF
THIS SURFACE IS QUITE DIFFERENT.
I WILL TELL YOU ABOUT THIS IN A
SECOND.
WHAT THIS LEADS TO IS THAT IN
PROTEOMIC STUDIES WE KNOW
THERE'S UPWARDS OF 100 PROTEINS
SPECIFIC TO LIPID DROPLETS
DEPENDING ON CELL TYPE, ET
CETERA.
THESE PROTEINS COME IN DIFFERENT
FLAVORS, SOME OF THEM YOU MIGHT
EXPECT SYNTHESIS OR DEGRADATION,
THAT MAKES SENSE.
THERE'S OTHERS WHICH YOU CAN
STILL RATIONALIZE SUCH AS
PHOSPHOLIPID REMODELING ENZYME
AND TRANSCRIPTION FACTORS THAT
WE START TO UNDERSTAND INVOLVED
IN REGULATION OF METABOLISM,
THERE'S ENZYMES INVOLVED IN
GLYCOSYLATION REACTIONS MANY
LABS FOUND AND NOBODY KNOWS WHAT
THEY ARE DOING AND WHY THAT
MATTERS.
IF YOU'RE INTERESTED WE BUILT A
KNOWLEDGE PORTAL WITH THE BROAD
INSTITUTE THAT ALLOWS YOU TO
BROWSE THE INFORMATION THAT AT
LEAST WE AND SOME OTHER LABS
HAVE GENERATED ON THE LIPID
DROPLETS.
HOW DO ALL THESE PROTEINS GET
THERE?
AS I MENTIONED WE DON'T REALLY
KNOW ANY OF THE PATHS.
SO WHAT WE HAVE LEARNED SO FAR
IS THAT GENERALLY THERE APPEAR
TO BE TWO PATHWAYS, ONE PATHWAY
YOU MAKE PROTEINS IN THE CYTOSOL
AND DIRECTLY BIND THE SURFACE.
IN A SECOND PATHWAY, PROTEINS
ARE INITIALLY INSERTED IN THE
E.R. AND GO TO THE SURFACE OF
LIPID DROPLETS.
IN THAT -- WE CALL THEM C YTOLS.
REGULATORS, TRANSCRIPTION
FACTORS, I'LL TELL YOU ABOUT ONE
OTHER PROTEIN LATER.
HOW DO THEY GET TO THE SURFACE
IS NON-TRIVIAL BECAUSE THEY HAVE
A SEQUENCE, A HELIX THAT HAS
PROPENSITY TO BIND THE MEMBRANE.
WHY DO THEY GO TO THIS RATHER
THAN THAT, THAT IS VASTLY MORE
ABUNDANT, MORE E.R. SURFACE THAN
LIPID DROPLETS IN CELLS, EVEN
THOUGH THEY HAVE THE SAME LIPID
COMPOSITION? PART OF THE
SOLUTION IS THE UNDERLYING
TRIGLYCERIDE CHANGES THE SURFACE
PROPERTIES OF THE MONO LAYER.
I DON'T HAVE TIME TO SHOW ALL
THE DATA BUT ESSENTIALLY IF YOU
IMAGINE A MEMBRANE HAS TO BE
TIGHTLY PACKED, NEXT TO EACH
OTHER.
IN A MONOLAYER YOU CAN PUSH THEM
APART A LITTLE BIT, THE
TRIGLYCERIDE CAN PUSH APART THE
CHAINS A LITTLE BIT, PACKING
DEFECTS, PROVIDES SURFACES THAT
CAN BE READ OUT BY PROTEINS.
THESE PACKING DEFECTS ARE MORE
ABUNDANT, MORE PERSIST ENT LARGR
ON THE SURFACE, MODELS WITH
TRIGLYCERIDES.
WHAT WE THINK HAPPENS FOR MANY
PROTEINS, I DON'T HAVE TIME TO
SHOW THE DATA, THEY HAVE
SEQUENCES THAT ARE DISORDERED IN
CYTOSOL, PROPENSITY TO BIND
THROUGH HYDROPHOBICS SUCH AS
TRYPTOPHANS, LEADING TO THE
FOLDING OF HELICES , LEAVING THS
PROCESS IRREVERSIBLE, REQUIRING
A DIFFERENT PROCESS TO GET THE
PROTEINS OFF THE SURFACE LATER
ON.
HOW ABOUT THAT OTHER PATHWAY?
SOME PROTEINS WE KNOW ARE
INSERTED INTO THE E.R., AND THEY
END UP ON THE LIPID DROPLET,
INCLUDING METABOLIC ENZYMES, G
PAT AND ASCL, INCLUDES THE RISK
FACTOR FOR DEVELOPMENT OF FATTY
LIVER DISEASE FROM HEPATITIS,
HSD17B13.
WHAT'S THE MECHANISM?
WE KNOW PROTEINS DON'T JUMP IN
AND OUT OF MEMBRANES, THIS CAN'T
BE IT.
INSTEAD WHAT WE FOUND IS THAT
WHEN YOU ANALYZE LIPID DROPLET
FORMATION, VISUALIZE RETICULUM
IN GREEN, ENZYMES IN RED AS CAR
GO, THEY START IN THE SAME CASE,
SAME PLACE, AND OVERLAP IN
COLOR.
WHEN YOU MAKE LIPID DROPLETS,
COLORS SEPARATE AND ENZYME ENDS
UP ON DROP DROPLET, ER STAYS B.
WE CAN DO LABELING WITH ANTIBODY
AGAINST ENDOGENOUS PROTEIN,
DECORATES THE SURFACE OF THE
LIPID DROPLET BUT NOT THE
ENDOPLASM RETICULUM CLOSE BY.
FIRST THOUGHT, IT'S SIMPLE,
WHAT'S THE BIG HOOPLA?
YOU START WITH A PROTEIN, MAKE
THE LIPID DROPLET, PROTEINS GOES
ON WHEN IT STARTS, RIGHT?
SOMETHING LIKE THAT.
DURING FORMATION PROTEIN GOES ON
THE LIPID DROPLET.
WELL, SOME PROTEINS CAN DO THIS
SUCH AS THIS LIVEDROP, YOU CAN
SEE DURING FORMATION THEY GO ON.
FOR THIS PARTICULAR PROBE WE
KNOW THIS IS DUE TO A CHANGE IN
CONFORMATION OF ENZYME THAT
ALTERS ITS ENERGY STATE BETWEEN
BILAYER AND MONOLAYER.
CURRENT MODEL WHEN THESE THINGS
ARE INSERTED INTO THE E.R.
MEMBRANE, THAT INSERTION
PLACES -- ANCHORS PROTEIN TO
BOTH SIDES OF THE MEMBRANE, WITH
THAT PLACES LARGE HYDROPHOBIC
TRYPTOPHANS IN THE MIDDLE OF A
BILAYER, HIGHLY ENUSUAL,
UNFAVORABLE, WHEN THEY COME TO
LIPID DROPLETS SWIM UP,
TRYPTOPHANS BECOME INTERFACIAL,
WE BELIEVE THAT DRIVES HOW THEY
ACCUMULATE.
I SHOULD SAY THIS IS ALL A MODEL
BECAUSE TO DATE THERE'S NOT YET
A STRUCTURE OF A LIPID DROPLET
PROTEIN.
TURNS OUT ONLY SOME PROTEINS CAN
DO THIS.
WE THOUGHT CASE CLOSED, DONE.
AS YOU FOLLOW ENDOGENOUS ENZYME
AND NOT THE FRAGMENT OFF IT
THERE'S A MYSTERY.
YOU CAN FOLLOW LIPID DROPLET
FORMATION FOR HOURS AND NOTHING
HAPPENS.
UNFORTUNATELY THIS PROJECTOR,
YOU CAN'T SEE THE GREEN DIFFUSE
BACKGROUND SIGNAL VERY WELL.
WHAT YOU CAN APPRECIATE IS THAT
AGAIN 350 MINUTES, NOTHING
HAPPENED.
WITHIN 10 MINUTES ONE OF THESE
DROPLETS BECAME ACCESSIBLE AND
TARGETS THAT ENZYME TO IT.
AND THEN VERY RAPIDLY LIKE IN
THE TRIGGERED FASHION THIS LIPID
DROPLET ACQUIRES THIS PROTEIN.
HOW DOES THAT WORK?
THE FIRST QUESTION IF IT WAS IN
THE E.R. WHY DOESN'T IT GET IN
HERE?
WELL, IT'S NOT HARD TO IMAGINE
THAT IF YOU HAVE THIS LARGE MEGA
DALTON COMPLEX AT THE BASE OF
FORMATION IT DOESN'T LET
EVERYTHING THROUGH, LARGER CARGO
CAN THEN GO THERE.
THIS IS THE CASE BECAUSE THE
EXPERIMENT I JUST SHOWED YOU
HERE SIMPLIFIED SAYS THAT, OKAY,
IN A WILDTYPE CELL AFTER HALF AN
HOUR EVEN THOUGH DROPLETS ARE
FORM ENZYME IS NOT THERE, LATER
IT IS, TAKE OUT SEIPIN, IT HOPS
ON.
THERE IS A PATHWAY BECAUSE AT
THE END THEY END UP ON LIPID
DROPLETS, THAT'S A REAL MYSTERY
FOR US.
IT LED US TO HYPOTHESIZE THERE
MUST BE TWO PATHWAYS, ONE EARLY
THAT ALLOWS THE ACCESS OF SOME
PROTEINS TO LIPID DROPLETS, AND
ONE LATER ONE THAT MAYBE USES
INDEPENDENT BRIDGES.
TURNS OUT AT LEAST IN TERMS OF
THE TIMING THERE'S A WHOLE SLEW
OF METABOLIC ENZYMES THAT APPEAR
TO USE THAT.
YOU MIGHT WONDER WHY SO
COMPLICATED, I'M HAPPY TO TALK
ABOUT THAT.
SO TURNS OUT REALLY THERE ARE
MULTIPLE MEMBRANE BRIDGES, THIS
IS AN EXPERIMENT, IMMEDIATE TIME
POINT.
THERE'S SIGNAL ALREADY ON THE
LIPID DROPLET.
AT THE BEGINNING OF THE
EXPERIMENT WE BLEACH THAT.
WHAT YOU CAN SEE THE SIGNAL
RECOVERS AND APPEARS TO RECOVER
THROUGH MULTIPLE CONNECTIONS
BETWEEN THE LIPID DROP LET
SURFACE AND ENDOPLASM RETICULUM
AND VISUALIZED BY
CRYOTOMOGRAPHY, YOU CAN SEE
E.R.s STUDDED WITH RIBOSOME,
THIS IS COLORED FOR BETTER
IMAGINATION.
THAT TRIGGERS HOW WOULD YOU
GENERATE SUCH MEMBRANE BRIDGES
BETWEEN THE E.R. AND LIPID
DROPLET INDEPENDENT OF THAT
INITIAL FORMATION SITE?
WE BANGED OUR HEAD FOR MANY
YEARS FRANKLY AND HAVE A NUMBER
OF IDEAS.
EVENTUALLY DECIDED TO DO A
VISUAL SCREEN KNOCKING OUT ONE
GENE AT A TIME, EVERY GENE IN
THE GENOME, ASKING WHICH ONES
SPECIFICALLY ABOLISH THE
RECRUITMENT OF LIPID DROPLET
PROTEINS TO THIS BUT NOT OTHER
CARGO, LIPID DROPLETS OR OTHER
SECRETORY PATHWAYS, AND FOUND A
CLASS OF TETHERING COMPONENTS,
MEMBRANE FUSION MACHINERY, AND
RECYCLING OF THAT MEMBRANE
FUSION THAT'S SPECIFICALLY
REQUIRED FOR THIS PATHWAY.
WE CAN SEE THOSE COMPONENTS NOT
ONLY ARE REQUIRED BUT THEY ARE
AT THE RIGHT TIME BECAUSE WE CAN
DO A SOMEWHAT COMPLICATED
EXPERIMENT HERE BUT JUST TO SHOW
WITHOUT EXPLAIN IN DETAIL, HAPPY
TO ANSWER QUESTIONS BIT, BUT WE
SET UP A SYSTEM WHERE YOU CAN
BASICALLY TEMPORARILY SEGREGATE
TRAFFICKING FROM FORMATION, AND
WHAT WE FOUND AS SOON AS WE
BASICALLY TRIGGERED, THESE ARE
MINUTES IN A CELL FUSION
EXPERIMENT, THE POTENTIAL FOR
THE PROTEIN TO GO ON TO LIPID
DROPLET, RIGHT BEFORE THAT, THE
PROTEIN WILL END UP ON THE LIPID
DROPLET, WE SEE THE MACHINERY WE
DISCOVERED, THIS IS ONE EXAMPLE,
WILL SHOW UP RIGHT AT WHAT WILL
BECOME A MEMBRANE FUSION SITE
BETWEEN THOSE TWO.
OUR BEST MODEL RIGHT NOW IS THAT
THERE IS AN INDEPENDENT
MACHINERY THAT INVOLVES E.R.
EXIT SITES AND MEMBRANE TETHERS
THAT ALLOWS FORMATION OF
SECONDARY CONTACT OR NOT
ACTUALLY CONTACT, MEMBRANE
BRIDGES BETWEEN THE E.R. AND
LIPID DROPLETS.
IF YOU THINK ABOUT IT, OF
COURSE, THIS MAY MAKE SOME
SENSE, ALLOWS YOU TO UNCOUPLE
PROTEIN COUPLING, THERE ARE MANY
CELL TYPES THAT DON'T MAKE A LOT
OF LIPID DROPLETS BUT STILL HAVE
TO CHANGE PROTEOME, MAYBE THIS
IS A PATHWAY REQUIRED FOR THAT.
HOWEVER, THERE'S MORE OPEN
QUESTIONS THAN ANSWERS HERE.
SO WE DON'T UNDERSTAND REALLY
HOW THIS BARRIER WORKS.
I CAN TELL YOU THE SIZE DOESN'T
MATTER.
IT'S NOT THE SINGLE
DISTINGUISHER HERE.
WE DON'T KNOW HOW THIS PATHWAY
HERE IS TRIGGERED AT ALL.
WE DON'T KNOW HOW PERSISTENT
BRIDGES ARE, WHETHER THEY ARE
ONE WAY OR TWO-WAY STREETS.
AND HOW THEY PARTICIPATE IN
PHYSIOLOGY.
SO WE'RE STILL BUSY FOR A WHILE.
WITH THIS I WANT TO SWITCH GEARS
FOR A SECOND HERE.
I TOLD YOU HOW TO MAKE A LIPID
DROPLET, GET THE PROTEINS ON
THAT MATTER.
ALL OF THIS OF COURSE PRESUMABLY
ONLY MATTERS IN CONTEXT WHERE
YOU ALSO HAVE MECHANISMS TO
MOBILIZE FATTY ACIDS, YOU WANT
TO STORE, PUT THINGS IN THE
BANK, YOU NEED TO WITHDRAW AT
LATER TIME POINT.
NOW, GENERALLY THERE ARE TWO
DISTRICT PATHWAYS HOW THIS CAN
BE ACHIEVED IN ALL THE CELLS
THAT PEOPLE HAVE LOOKED AT.
THE FIRST ONE IS THAT ENZYMES
SUCH AS APGL THAT SHOWED UP ON
MY SLIDES ALREADY CAN TARGET THE
SURFACE OF LIPID DROPLETS AND
ESSENTIALLY HYDROLASES THAT
CLEAVE ESTER BONDS, PARTICULARLY
THE FIRST ONE, HYDROLYZE
TRIGLYCERIDE AND RELEASE FATTY
ACID, THREE PACES TAKE THE
FIRST, SECOND, THIRD ACEEL
CHANGE, DISCOVERED BY RUDY
TECHNER, AND WHO HAS DONE
BEAUTIFUL WORK SHOWING HOW THIS
WORKS, PARTICULARLY IN
ADIPOCYTE, ESSENTIALLY NOT
UNDERSTOOD IN ANY OTHER CELL
TYPE.
THERE'S A SECOND PATHWAY THAT
YOU CAN IMAGINE WHICH WORKS
DIFFERENT, WHERE A LIPID DROPLET
OR PART OF A LIPID DROPLET IS
INTERNALIZED INTO AN
AUTOPHAGOSOME STRUCTURE, ACID
LIPase, THIS LIPO PHAGEY WAS
MORE THAN TEN YEARS AGO,
REPORTED IF YOU KNOCK OUT
AUTOPHAGY IN LIVER YOU
ACCUMULATE A LOT OF LIPID
DROPLETS.
THERE'S HUNDREDS OF PAPERS AFTER
THAT.
IT'S BEEN REALLY DIFFICULT TO
ASK, OKAY, HOW MUCH HAPPENS, HOW
DOES IT WORK AND REGULATE AND TO
ME AT LEAST MOST IMPORTANTLY HOW
DOES THIS CONTRIBUTE TO
PHYSIOLOGY.
BECAUSE THE ONLY TOOLS THAT WE
HAVE WERE BLUNT LIKE WE KNOCK
OUT AUTOPHAGY BUT NOTHING HAS
BEEN KNOWN SPECIFICALLY HOW DO
YOU CONNECT THE LIPID DROPLETS
TO THE AUTOPHAGESOME, AN
INTERESTING QUESTION.
LUCKILY ALSO TO BOB AND CHUNG.
THE DISCOVERY WAS SERENDIPITOUS,
STARTED WITH ASKING RANDOMLY FOR
LIPID DROPLET PROTEIN WHERE IS
IT LOCALIZED?
THIS PROTEIN IS CALLED SPARTAN.
WHAT WAS PECULIAR WAS THAT WHEN
WE LOCALIZE WITH A KNOCK-IN
ENDOGENOUS LOCUS WE FOUND THAT
IT LOCALIZED TO LIPID DROPLETS,
BECAUSE THEY STAIN WITH NEUTRAL
LIPID DYES, BUT DOESN'T LOCALIZE
TO THE SAME LIPID DROPLETS AS A
SURFACE MARKER PLIN3.
PARTICULARLY WE ASKED WHAT'S
DIFFERENT ABOUT THESE LIPID
DROPLETS THAN THE REST OF THE
LIPID DROPLETS?
WHAT HE DID WAS CO-LOCALIZED
WHAT MIGHT BE DIFFERENT AND
FOUND QUICKLY THAT THE SPARTIN
DROPLETS LOCALIZE FLEX TO A
LYSOSOME, AND ARE ALMOST ALWAYS
MARKED WITH AN LC3 PROTEIN, AND
CORE PART OF THE AUTOPHAGY
MACHINERY.
AND I WILL TELL YOU ABOUT
SPARTIN NOW.
WHAT IS THIS PROTEIN?
IT IS A PROTEIN THAT HAS A
COUPLE OF SUGGESTED DOMAINS.
THERE'S SOMETHING IN THE
N-TERMINUS THAT'S CALLED A
MICROTUBULE INTERACTING DOMAIN
WHICH MOSTLY LIKELY DOES NOT
INTERACT WITH TUBULES.
THE YOUR -- THERE'S A UBIQUITID
SENESCENCE DOMAIN, ASSOCIATED
THROUGH COMPLEX PHENOTYPE.
WHAT WE POSTULATED IS THAT MAYBE
THIS PROTEIN SERVES OR
HYPOTHESIZED AS A PROTEIN THAT
BRIDGES THE LIPID DROPLET AND
LYSOSOME OR AUTOPHAGIESOME.
I DON'T HAVE TIME TO SHOW THE
DATA BUT WHAT WE FOUND IS THAT
THE SENESCENCE DOMAIN IS
COMPOSED OF A SERIES OF REPEATED
HELICES, YOU KNOW, THERE'S A
WHOLE SET OF THEM IN HERE.
WHAT WE FOUND IS THAT THESE
PROTEINS ARE IN CELLS SUFFICIENT
TO TARGET A PROTEIN TO LIPID
DROPLETS, CONSISTENT WITH WORK
FROM OTHERS THAT WAS PUBLISHED
BEFORE.
IN ADDITION, THIS DOMAIN IN THE
MIDDLE REALLY LOOKS LIKE A PH
DOMAIN, AND CAN INTERACT IN AN
UNCANONICAL MOTIF WITH LC3.
AND WE'VE SHOWN DIRECTLY, I
DON'T HAVE TIME TO SHOW ALL OF
THIS, THAT THIS PROTEIN BINDS
DIRECTLY IN VITRO.
BASED ON THIS WE THINK THIS
PROTEIN SPARTIN MIGHT BE
FUNCTIONING AS RECEPTOR THAT
BRIDGES THE LIPID DROPLET
SURFACE TO THE AUTOPHAGYIC
MACHINERY.
AND AGAIN, THE PAPER WILL BE
PUBLISHED SHORTLY SO YOU CAN
HAVE A LOOK AT THAT.
BUT THIS BRINGS UP THE QUESTION
PROTEIN-PROTEIN INTERACTIONS ARE
NICE BUT WHAT DO THEY DO.
WE FUSED A FLUOROPHORE TO A
PROTEIN, PECULIAR FLUOROPHORE IN
AS MUCH AS IT HAS A DIFFERENT
EXCITATION MAXIMUM IN NEUTRAL
AND ACIDIC PHs, YOU CAN
DISTINGUISH IN THE CYTOSOL.
IT WILL BE -- HAS A GREEN
MAXIMUM, IF IT GOES TO ACIDIC
ENVIRONMENT SHIFTS TO RED
MAXIMUM, TRANSITION YOU WOULD
EXPECT FROM SOMETHING BEING
DELIVERED TO A LYSOSOME.
, FROM GREEN TO RED HERE.
WHAT WE FOUND, IF YOU TAKE
CELLS, LOAD WITH FATTY ACID,
WITHDRAW THE FATTY AS I.D. TO
FORCE THEM TO MOBILIZE STORES,
APPEARS TO DELIVER TO
COMPARTMENT BLUNTED WITHOUT
SPARTIN.
THIS IS A CRISPR KNOCKOUT.
CELLS THAT DO NOT HAVE SPARTIN
ACCUMULATE TRIGLYCERIDE BECAUSE
NOW THEY CAN'T MOBILIZE AS WELL.
THIS ACCUMULATION IS NOT DUE TO
DIFFERENCE IN SYNTHESIS BUT
DEGRADATION, ROUGHLY ON THE SAME
SCALE THAT YOU OBSERVED WITH
DEFECT, THAT YOU OBSERVE WITH
THE MUTATION OF AUTOPHAGY
MACHINERY THAT'S NOT ON THE
SLIDE, IF YOU MUTATE BOTH
TOGETHER THERE'S NO ADDITIONAL
PHENOTYPE.
WITH ALL OF THIS THE CURRENT
WORKING MODEL THAT WE HAVE IS
THAT SPARTIN DECORATES THE
SURFACE AND INTERACTS DIRECTLY
WITH LC3 AND DELIVERS THE
PROTEIN -- DELIVER LIPID
DROPLETS TO LYSOSOME FOR
DEGRADATION A LAYINGS, NOW
-- FOR
DEGRADATION.
WHY IS THIS CALLED SPARTIN?
IT'S ALSO KNOWN AS SPG20, FOUND
IN HUMAN MUTATIONS THAT CAUSE A
COMPLEX SPASTIC PARAPLEGIA,
TROYER SYNDROME.
THAT IS CHARACTERIZED BY
AUTOSOMAL PARAPLEGIA WITH MUSCLE
WEAKNESS, SMALL STATURE AND
NEUROLOGICAL DEFECTS, SPREAD ALL
OVER THE PROTEIN QUITE FRANKLY
BUT CONCENTRATE ALSO TO SOME
DEGREE ON THE MIDDLE DOMAIN AND
SOMEWHAT IN THE SENESCENCE
DOMAIN.
CONSISTENT WITH THIS IDEA,
SOMEHOW HAS SOMETHING TO DO WITH
NEURONS, HIGHLY EXPRESSED IN THE
BRAIN AND PARTICULARLY IN
NEURONS.
FRANKLY, THIS IS THE LAST PLACE
I SUSPECTED LIPID DROPLETS TO
MATTER.
IF YOU THINK ABOUT THE BRAIN,
NOT NECESSARILY THE FIRST PLACE
ADIPOSE TISSUE DELIVERED, THAT'S
WHERE PEOPLE LOOK, WHAT ABOUT
THE BRAIN?
WE MADE INITIALLY AS A FIRST
EXPERIMENT MADE NEURONS IN VITRO
FROM iPS CELLS, DIFFERENTIATE
EITHER WILD TYPE OR SPARTIN
KNOCKOUTS AND FOUND THAT ONLY
WHEN YOU DON'T HAVE SPARTIN YOU
APPEAR TO ACCUMULATE LIPID
DROPLETS IN THE CELL BODY.
THIS IS CELL CULTURE, I MOTOR
NEURONS ARE MAYBE A MODEL OF
NEURONS BUT ARE NOT IN THE
BRAIN.
WE DEVELOPED A SYSTEM TO TEST
THIS IN VIVO AND SPECIFICALLY
WHAT WE DID WAS PARK AT WAYNE
STATE DEVELOPED A SYSTEM WHERE
WE INJECT EITHER CONTROL OR
DOMINANT NEGATIVE FRAGMENT OF
SPARTIN WE DEVELOPED IN VITRO
AND ASKED EFFECT, EXPRESSED ONE
ON ONE SIDE OF THE MOUSE BRAIN,
THE OTHER ON THE OTHER SIDE OF
THE MOUSE BRAIN.
YOU CAN SEE CONTROL EXPRESSES
MUCH HIGHER, WE ASKED WHAT ARE
THE CONSEQUENCES.
WE FOUND THAT SPECIFICALLY CELLS
THAT EXPRESS THIS DOMINANT
NEGATIVE SPARTIN START TO
ACCUMULATE LIPID DROPLETS IN
THEIR CELL BODY.
AND THEN WHEN WE GO AND CUT OUT
THOSE REGIONS SPECIFICALLY WE
CAN SEE THAT THE BRAIN OF THOSE
ACCUMULATES TRIGLYCERIDE AND DAG
BUT THE REST OF THE LIPIDS ARE
NOT CHANGED.
WE THINK FOR SOME REASON
AUTOPHAGY APPEARS TO MATTER IN
THE NEURONS, REQUIRED FOR NORMAL
NEURONAL FUNCTION.
THAT'S SURPRISING, AND I DON'T
KNOW WHY YET.
WHAT IS TANTALIZING IS THAT WHEN
WE GO BACK WHAT I HAVEN'T TOLD
YOU YET THERE ARE MUTATIONS IN
SEIPIN, SPG17, THEY ARE
DIFFERENT MUTATIONS OF THE
SEIPIN THAT DON'T LEAD TO
LIPODYSTROPHY, MISSENSE
MUTATIONS MOSTLY.
THEY LEAD TO SPASTIC PARAPLEGIA
SPARTIN I TOLD YOU ABOUT.
CONCOMITANTLY DDHD2 IS A
TRIGLYCERIDE HYDROLYZED THAT'S
BRAIN SPECIFIC, AND THAT
MUTATION -- SO MUTATIONS IS
LEADING TO SPASTIC PARAPLEGIA.
THERE'S A SLEW OF MUTATIONS THAT
ARE KNOWN TO GIVE YOU SPASTIC
PARAPLEGIA, OFTEN AFFECT E.R.
FUNCTION AND LIPID DROPLETS.
WE'RE NOW QUITE INTERESTED TO
ASK WHAT MIGHT BE THE FUNCTION,
PARTICULARLY IN MOTOR NEURONS.
I'M HAPPY TO TALK BIT, CAN'T
ANSWER THE QUESTION TODAY.
WHAT I TOLD YOU TODAY, SOME
HIGHLIGHTS, I HOPE I HELPED YOU
UNDERSTAND A LITTLE BIT HOW WE
CURRENTLY THINK TRIGLYCERIDE
SYNTHESIS OCCURS IN THE DGAT
ENZYMES, THERE'S DEDICATED
MACHINERY THAT FORMS A VERY
LARGE MEGA DALTON COMPLEX THAT
ALLOWS FORMATION OF LIPID
DROPLETS OUT OF THE E.R., THERE
BY PROTECTING THE CELL FROM
LIPOTOXICITY OF DISTRIBUTED
LIPID INTERMEDIATES.
THERE ARE DISTINCT PATHWAYS TO
GET ONTO LIPID DROPLETS, SOME WE
START TO UNDERSTAND, TO SOME
DEGREE.
AND THAT THERE'S A FASCINATING
NEW AREA OF LIPID DROPLET
BIOLOGY WITH AUTOPHAGY IN THE
BRAIN WHERE WE START TO HAVE
MOLECULAR PLAYERS THAT ALLOW US
HOPEFULLY TO ANALYZE THE
PHYSIOLOGY OF ALL THESE
PROCESSES.
AND WITH THIS, I WANT TO
PARTICULARLY THANK THE PEOPLE
WHO HAVE DONE THE WORK.
I HIGHLIGHTED WORK BY CHUNG, A
FANTASTIC POSTDOC.
WHEN YOU'RE SUCCESSFUL AS A
POSTDOC YOU GO SOMEWHERE ELSE.
YOUR MENTOR STAYS.
IN OUR CASE SHE GOT A JOB AT
HARVARD, SHE STATE, WE LEFT.
SHE'S GREAT.
WORK ON MEMBRANE TRAFFICKING, A
FANTASTIC MD/PHD STUDENT,
STRUCTURAL BIOLOGY A DGATs AND
WORK ON SEIPIN, NOW FACULTY IN
TEXAS AND ARLT IS AN OUTSTANDING
BIOCHEMIST AT THE DANA-FARBER
WHO WORKED ON THE SEIPIN
STRUCTURE.
I WANT TO THANK OUR FUNDING,
PARTICULARLY NIH.
THANK YOU FOR SUPPORT OF OVER
MANY YEARS, PARTICULARLY NIGMS,
ALSO HHMI WITH THAT ALL TAKE ANY
QUESTIONS YOU MIGHT HAVE.
AND THE BLUEFIELD.
>> PLEASE USE THE MICROPHONES.
AND I'LL ANNOUNCE FIRST CME
CODE, IF I REMEMBER CORRECTLY
44463.
AND I GUESS THAT'S IT FOR NOW.
PLEASE.
>> VERY NICE PRESENTATION.
SO TWO-PART QUESTION.
THE SPARTIN PROTEIN IS THAT
SPECIFIC ONLY FOR LIPID DROPLETS
TO DELIVER THOSE TO THE
AUTOPHAGOSOME OR DO YOU FIND IT
ON MAYBE OTHER COMPARTMENTS OR
ORGANELLES?
>> YEAH, THANKS FOR THAT
QUESTION.
I SHOULD HAVE SAID THIS PROTEIN
WAS ALSO REPORTED IN A NUMBER OF
OTHER CONTEXTS.
FOR INSTANCE, IN THE CLEAVAGE OF
MITOTIC CELLS, WE TAG THE LOCUS
AND NEVER SEE IT OTHER THAN
DISTRIBUTED IN THE CELLS OR AT
LIPID DROPLETS CONSISTED WITH
WORK AT LOCALIZATION, ALSO
PROTEOMICS.
>> WHAT IS THE SIGNAL FOR
LOCALIZING TO THE LIPID
DROPLETS?
IS IT LIKE BY DEFAULT SITTING
THERE AND, YOU KNOW, TURN
ON/OFF?
>> YOU CAUGHT ME, RIGHT?
I SET THIS UP NICELY, WOULDN'T
IT BE NICE IF WE COULD
UNDERSTAND THE REGULATION,
THAT'S HOW THE REGULATION WORKS.
I DON'T KNOW.
IT IS -- YOU CAN SPECULATE
THERE'S A MONO YOUR BIC
WILLATION SITE EXTENDED TO POLY
UBIQUITIN CHAIN, THAT APPEARS TO
BE REGULATED BY THE METABOLIC
STATE.
AND CORRELATES WITH A CHANGE IN
LOCALIZATION.
WE DON'T KNOW HOW THAT WORKS,
WHAT TRIGGERS.
THERE'S CERTAIN STIMULI BUT ONES
IN VITRO ARE NOT PHYSIOLOGIC.
YOU DON'T GO AND WITHDRAW FATTY
ACIDS, SO WE DON'T KNOW IS THE
SHORT ANSWER ABOUT IT APPEARS
REGULATED.
I DON'T KNOW, THIS PROTEIN
APPEARS TO BIND SUBSET OF LIPID
DROPLETS, BUT THE SIGNAL YOU GET
IS ALL AROUND.
LIKE ONE PROTEIN BINDS, THERE
MUST BE SOMETHING DIFFERENT
ABOUT THESE DROPLETS, RIGHT?
WHY DO THIS ALL GO TO THAT
DROPLET AND NOT THE ONE NEXT TO
IT, AND I DON'T KNOW THAT
EITHER.
>> YOU MENTIONED TRANSFERASES,
AND PLASMA MEMBRANE, MUCH MORE
THAN ENDOPLASMATIC, IF YOU
EXTEND THAT TRANSPORTER CELLS,
THE PLASMA IN THE BRAIN, THE
THICKNESS OF THESE PLASMA
MEMBRANES, COMPOSITION VERSUS
ENDOPLASMIC RETICULUM, ARE
PROTEINS EMBEDDED HERE?
>> IF I UNDERSTAND YOUR
QUESTION CORRECTLY, YOU'RE
TALKING ABOUT A CONCEPT THAT'S
ALSO KNOWN AS HYDROPHOBIC
MISMATCH, THE LENGTH OF A
CHANNEL MEMBRANE DOMAIN IS
MATCHED TO NICKNESS OF --
THICKNESS OF MEMBRANE,
INCREASING TOWARDS THE PLASMA
MEMBRANE, RIGHT?
IF THAT'S MATCHED.
IF YOU THINK BIT, LIPID DROPLET
IS THE EXTREME CASE OF A THICK
MEMBRANE.
YOU HAVE ONE SET OF
PHOSPHOLIPIDS, AND THE OCEAN
UNDER IT ESSENTIALLY.
WE DO THINK THAT MATTERS FOR
THAT MECHANISM BUT IT IS
IMPORTANT TO REMEMBER YOU CAN'T
PRESUMABLY HAVE TRANSMEMBRANE
DOMAIN, PER SE.
THE TRANS PART, NO OTHER LUMEN,
THEY ALL DIP.
THAT MAKES IT FUNDAMENTALLY
SOMEWHAT DIFFERENT.
>> PROTEIN TRANSFERASE
N-TERMINAL END IS TRANSMEMBRANE
DOMAIN.
IF YOU EXTEND, THE PROTEIN WOULD
GO TO PLASMA MEMBRANE.
>> I'M AWARE OF THAT.
I DO NOT KNOW WHETHER THAT PLAYS
A ROLE HERE.
I DID MENTION BECAUSE -- I
MENTIONED THIS EVERY ONCE IN A
WHILE.
SOMEONE WILL HAVE A CLUE AT SOME
POINT.
IF YOU JUST LOOK THROUGH
LITERALLY NOW HUNDREDS OF
PROTEOMIC STUDIESU GRIND YOUR
CELLS, PUT THE DROPLETS ON TOP,
MASS SPECTROMETRY, IT TELLS YOU
MORE ABOUT MASS SPECTROMETRY,
BUT THERE'S LOTS OF OTHER
THINGS.
IN HUNDREDS OF STUDIES ONE OF
THE THINGS THAT REPEATEDLY COMES
UP IS GLYCOSYLATION MACHINERY
LOCALIZED BY MICROSCOPY.
TO THIS DAY I HAVE ZERO IDEA WHY
THEY ARE THERE.
YOU CAN SPECULATE, THINK ABOUT
THE STRUCTURAL, RECEPTOR IN
LIPID DROPLETS BUT NOBODY KNOWS.
THAT'S A LONG WINDING ANSWER FOR
I HAVE NO CLUE.
>> A QUESTION ONLINE BEFORE YOU
GO.
>> SURE.
>> SO IN YOUR PICTURE OF THE
ENCAPSULATED LD IN LYSOSOME, TWO
SURFACES SEEM TO CONTACT.
DOES THE SNARE ET CETERA
MACHINERY PLAY A ROLE?
WHY IS ENCAPSULATION LITHOPHAGEY
NEEDED?
>> SO WE DO NOT KNOW ANY OTHER
MACHINERY AT THIS POINT THAT'S
REQUIRED FOR SPECIFIC
LITHOPHAGY.
NORMALLY PHAGOSOME FORMS AND
RECUSES.
WE DON'T UNDERSTAND WHY.
THE ENCAPSULATION PRESUMABLY IS
NEEDED TO ALLOW FOR THE ACTION
OF ACID LIPASE.
THERE'S A PROTEIN CALLED LAO
ONLY WORKS IN ACIDIC PH,
KNOCKOUT MICE ARE IMPORTANT FOR
MOBILIZING TRIGLYCERIDE.
THEY DO NOT KNOW THE OTHER
MACHINERY THAT'S INVOLVED IN
THIS PROCESS YET.
>>CURVATURE OF LIPID DROPLETS
CHANGE A LOT FROM WHEN BORN TO
WHEN THEY ARE LARGE.
IS THERE EVIDENCE THAT PLAYS A
ROLE IN ACTIVATING ENZYMES THAT
SIT ON THE SURFACE OF THE LIPID
DROPLET?
>> SHORT ANSWER IS NO.
ACTUALLY THERE'S -- IF YOU GO TO
THE BIOCHEMISTRY OF THE PROCESS
THERE'S A LOT OF MYSTERY.
WE HAVE NOT WORKED ON THIS SO I
DON'T KNOW ABOUT BESIDES WHAT'S
PUBLISHED.
IF YOU THINK ABOUT FOR INSTANCE
APGL, THE LIPASE, SITS ON THE
SURFACE OF LIPID DROPLETS,
PERFECTLY CAPABLE OF DOING WHAT
IT DOES, BUT IT'S INACTIVE.
THERE'S ANOTHER PROTEIN THAT'S
ALSO ON THE SURFACE OF LIPID
DROPLETS, AND THAT NEEDS TO BIND
THAT PROTEIN TO ACTIVATE THE
LIPASE.
THEY ARE BOTH THERE, MASSIVE
CONCENTRATIONS, RIGHT?
BECAUSE -- BUT CAN'T INTERACT
BECAUSE THAT'S BINDING SOMETHING
ELSE.
ALL THESE THINGS ARE STILL IN
THE PHASE WHERE WE HAVE
ESSENTIALLY SOME VERY HIGH LEVEL
CONCEPTS BUT REALLY NOBODY
UNDERSTANDS, NO STRUCTURES FOR
THESE THINGS, BIOCHEMISTRY IS
REALLY NOT VERY WELL UNDERSTOOD.
AND DESPITE THAT THERE ARE DRUGS
FOR INSTANCE LIKE THE APLG
INHIBITORS PIONEERED FOR KAKEXIA
CANCER AND OTHER THINGS.
I HAVE NO ANSWER, HAVEN'T
EXPERIMENTED WITH THAT.
>> THANKS.
>> GREAT TALK, LOOKING AT DGAT1
AND 2, 1 HAVING LATERAL GATE DO
YOU THINK VERSUS CYTOSOLIC
ORIENTED SITE OF DGAT2 IS IT
ACCESSING FROM DIFFERENT
LEAFLETS, WHETHER IN THE
CYTOPLASMIC OR INNER LEAFLET?
>> I THINK THE GENERAL IDEA IN
THE FIELD IS THAT THEY CAN
FLIP-FLOP FAST BECAUSE THEY HAVE
ESSENTIALLY NO POLAR HEAD GROUP.
WHAT IS SPECIFIC ABOUT DGAT1, IT
TAKES FROM THE E.R.
WE'VE SHOWN, AN MANY OTHERS,
SOME IN CANADA, THAT DGAT2
LOCALIZES IS THE SURFACE OF
LIPID DROPLETS, WE DON'T DON'TN
DROPLET OR CONTACT SIDES,
TEMPTING TO THINK IT'S
DETOXIFYING BUT DGAT 2 LOCALLY
EXPANDS.
HORTON AT SOUTHWESTERN HAS
BEAUTIFUL WORK SUGGESTING
CHANNELED BETWEEN LIPOGENESIS TO
TRIGLYCERIDE SYNTHESIS BUT CELL
BIOLOGICAL BASIS IS NOT YET
KNOWN.
>> THAT LEADS INTO THE
FOLLOW-UP, TALKED ABOUT
TARGETING ENZYMES TO DROPLETS,
GPAT4 IS THE BASIS OF LIVEDROP
SENSOR, MAKING LISO LIPID.
DO YOU THINK THAT THEY ARE
PLAYING A ROLE?
EXIT SITES ARE ALSO ALLEGEDLY
ENROICHED IN LISO LIPIDS.
>> GOOD QUESTIONS AND
EMBARRASSING I DON'T HAVE
ANSWERS.
I CAN TELL YOU IF YOU JUST DO
CRUDE EXPERIMENTS AND PULL ON
GPATS AN CELLS YOU'LL FIND THE
EKPAT.
PULLING ON DGAT YOU CAN FIND
THINGS THAT DESATURATE.
THERE'S A FAIRLY HIGH CHANCE,
SOME PEOPLE WHO JAY HORTON
PARTICULARLY, BEAUTIFUL WORK ON
THE FATTY ACID SYNTHASE COMING
TOGETHER ON A COMPLEX AROUND
LIPID DROPLETS, THERE'S A FAIRLY
HIGH CHANCE THERE'S SOME SORT OF
CELLULAR ORGANIZATION OF THIS
PATHWAY.
BUT IT'S JUST A CHANCE.
HONESTLY THIS FIELD HASN'T BEEN
NECESSARILY A CENTER OF
ATTENTION FOR CELL BIOLOGY FOR
MANY YEARS, AND THEN THE TOOLS
ARE ONLY NOW CATCHING UP.
IF YOU OVEREXPRESS THESE
ENZYMES, HELL BREAKS LOOSE AND
EVERYTHING IS EVERYWHERE.
VERY HARD TO STUDY.
>> THANK YOU SO MUCH.
>> TRY TO MAKE IT QUICK.
SURFACE OF THE LIPID DROPLETS,
BECAUSE OF NON-CANONICAL NATURE,
I WOULD IMAGINE THE PROTEINS
THAT BIND AND PROTEIN STRUCTURES
ARE ALSO NON-CANONICAL.
CURIOUS FOR EXAMPLE TWO LIKE
ALPHA FOLD BASES ON TRAINING
SETS PROBABLY RATHER
UNREPRESENTED IN WHAT YOU NEED
TO PREDICT STRUCTURES AT
PROTEINS.
CAN YOU SHARE YOUR THOUGHTS
ABOUT THAT PROBLEM?
>> YEAH, THERE ARE NO PUBLICLY
AVAILABLE STRUCTURES FOR A FULL
LENGTH LIPID DROPLET PROTEIN.
THERE'S BEEN SOME BEAUTIFUL WORK
ON LIPOPROTEINS, THE OTHER LIPID
DROPLETS, MUCH OLDER AND BETTER
STUDIED BUT NOT THREE
DIMENSIONAL STRUCTURES, MORE
LIKE HOW DOES THIS WORK?
YEAH, BECAUSE OF THAT WE DON'T
FIND ALPHAFOLD AS VALUABLE FOR
THIS PROTEIN AS WE FIND FOR
OTHER THINGS, E.R. PROTEINS,
WOW, THIS IS AMAZING, AND THEN
WE GET CryoEM MAP AND JUST
LOOKS REALLY BLOWN AWAY BY
POTENTIAL.
WE'VE NOT HAD THAT EXPERIENCE
WITH LIPID DROPLET PROTEINS
WHERE OFTEN YOU GET SOMETHING
THAT LOOKS LIKE A RANDOM
COIL-ISH THING WITH A BUNCH OF
HELICES LEFT OR RIGHT, OR WHERE
IT'S HARD TO MAKE SENSE.
>> THANK YOU.
>> I DON'T THINK IT'S THAT
USEFUL YET.
>> THANK YOU.
WE DO HAVE SOME OTHER GREAT
QUESTIONS BUT WE'LL GET THEM TO
YOU ELSEWHERE FROM OUR
COLLEAGUES.
YES?
>> [OFF MICROPHONE].
>> NOT YET.
>> I WANT TO THANK YOU AGAIN.
[APPLAUSE]