WELCOME, EVERYONE, TO TODAY'S
SESSION OF THE WEDNESDAY
AFTERNOON LECTURE SERIES HERE AT
THE NIH.
I AM A SENIOR INVESTIGATOR IN
THE INTRAMURAL PROGRAMME OF
NICHD AND TODAY'S LECTURE IS
HOSTED BY THE MEDALS IN BIOLOGY
SCIENTIFIC INTEREST GROUP.
IT'S A GREAT HONOUR AND PLEASURE
TO INTRODUCE TODAY'S SPEAKERS
WHO IS OUR NEXT DOOR NEIGHBOUR,
PROFESSOR IQBAL HAMZA AT THE
CENTRE OF BLOOD OXYGEN TRANSPORT
AT THE MARYLAND SCHOOL OF
MEDICINE.
IQBAL GREW UP IN INDIA, IN BOR
BAY,BOMBAY AND RECEIVED HIS DEGS
IN BIO CHEMISTRY FROM THE
UNIVERSITY OF BOMBAY.
HE THEN CAME OVER TO THE U.S. TO
THE STATE UNIVERSITY OF
INTERSECT BUFFALO CAMPUS IN THE
LAUGH OF MARK BRIAN, WHERE HIS
WORK WAS THE ROLE OF BACTERIAL
IRON RESPONSE REGULATE OR IN
COORDINATING IRON HOMEOSTASIS
AND HEME SYNTHESIS ASK THIS IS
WHEN THE BUG OF METALS BIOLOGY
BIT IQBAL BECAUSE HE WENT ON TO
DO A POSTDOCTORAL RESEARCH WHERE
HE EXPANDED HIS TWO CHAIRS TO
TRANSGENIC MICE TO MODEL HUMAN
GENETIC DISEASE OF COPPER MET
BOLISM AND WILL SONS AND
ESTABLISHED THE MOLECULAR FOR
COPPER TRAFFICKING BY SHAPER OWN
AND IN HIS OWN WORDS WITH A
THREE-WEEK CRASH COURSE AT THE
COLD SPRING HARBOUR LAB, HE
STARTED AT THE UNIVERSITY OF
MARYLAND WHERE HE EMBARKED
OBJECT A COMPLETELY NEW
DIRECTION THAT OF HIM
TRAFFICKING PATHWAY IN YOU
CHARIOT SO THERE ARE NUGGETS AND
LOOKING TO START YOUR LAB.
HIS CHOICE WAS PRECISE BECAUSE
IT TURNS OUT THAT IT DOES NOT
UTILIZE HEME BUT ACTUALLY
UTILIZES ENVIRONMENT, IT DOES
NOT MAKE ITS OWN HEME BUT
UTILIZES ENVIRONMENTAL HEME AND
WITH THE SYSTEMIC LAB, THEY HAVE
GONE ON TO MAKE PIONEERING
DISCOVERIES IN TRAFFICKING AND
ESTABLISHED CONNECTIONS TO A
NUMBER OF HUMAN DISEASES SOME OF
WHICH I HOPE HE TOUCHED ON
TODAY.
HE HAS RECEIVED NUMEROUS AWARDS
INCLUDING THE NIH RESEARCH
CARRIER DEVELOPMENT AWARD AND
THE JUNE FACULTY EXCELLENCE
AWARD AND THE FACULTY RESEARCH
AND SCHOLARSHIP PRICE AT THE
UNIVERSITY OF MARYLAND AND HE IS
A FELLOW OF THE JAPAN SOCIETY OF
PROMOTION OF SCIENCE AND A
FELLOW OF THE AMERICAN
ASSOCIATION FOR THE ADVANCEMENT
OF SCIENCE.
BUT MORE IMPORTANTLY, HE HAS
BEEN A PARTICULARLY STRONG
MENTOR OF YOUNG SCIENTISTS, IN
THE AREA OF METAL BIOLOGY, NOT
ONLY IN HIS OWN DEPARTMENT BUT
OUTSIDE IN THE COMMUNITY.
AND IT'S REALLY VERY HIGHLY
APPRECIATED BECAUSE OF THAT.
HE HAS HAD A LONG INTERACTION
WITH THE NIH INTRAMURAL AND WE
LOOK FORWARD TO CONTINUING
INTERACTIONS AND THANK YOU VERY
MUCH FOR ACCEPTING OUR
INVITATION, IQBAL.
>>IT'S REALLY AN HONOUR AND A
PLEASURE TO BE HERE.
IT'S NOT THE FACT I'M AT NIH
AND IT IS AN HONOUR HALF OF THIS
AUDIENCE, RIGHT, AND THE JOKE
ASIDE, ONE OF THE BEST THINGS OF
BEING AT MARYLAND IS
THE FACT THAT IT'S SO CLOSE TO
THE NIH AND I HAVE SOME I CAN
CAN COLLABORATE WITH THEM AND
SOME OF THEM ARE IN THE AUDIENCE
AND WHAT IS NOT CLEAR FROM THE
PRODUCTION IS THAT TWO OF MY SA
BAT CALECHES ARE 2010 IN 2019 AT
THE NIH THE FIRST ONE WITH AND
THE SECOND ONE WITH DAVID
BODINE AND YOU WILL ALSO SEE
THERE'S A THEME AND THE TALK
TODAY AND THAT IS THE POWER OF
USING MODEL SYSTEMS TO UNCOVER
UNANSWERED QUESTIONS AND YOU
WILL SEE THAT AND THE USE OF
THOSE ORGANISMS SHOWN HERE AS
ELEGANS SO OUR FUNDAMENTAL
QUESTIONS ARE TO UNDERSTAND THE
BIOLOGY OF HEME.
I DON'T FIT IN AS A
MICROBIOLOGIST, HEMATOLOGIST, A
FUNDAMENTAL QUESTION IS SIMPLE,
HOW DOES HEME, AS YOU WILL SEE
IN THE NEXT SLIDE, HYDRO PHOBIC,
SER A TOXIC, SICK LICK, ORGANIC
RING MOVES AROUND BETWEEN CELLS
AND WITHIN CELLS.
SO ANSWER THAT WE USE ELEGANS
AND TO BE ABLE TO UNCON THESE
PATHWAYS AND IN HUMAN HEALTH AND
DISEASE AND WE HAVE TO CROSS
MANY DIFFERENT BARRIERS AND WE
USE MANY DIFFERENT MODEL SYSTEMS
AND I DID MY PHD DOING BACTERIAL
GENETICS SO FROM BACTERIA TO MAN
OR TO MOUSE, THERE ARE OTHER
ORGANISMS THAT YOU CAN TRANSCEND
AND EACH ORGANISM THAT YOU USE
CAN BE EXPLOITED FOR ITS PURPOSE
IT BRINGS TO TH TO THE GENETIC
TOOLBOX.
WE USE ZEBRAFISH AND SO WE HAVE
A LARGE MOUSE COLONY AND
SOMETHING THAT I DID NOT
ANTICIPATE 20 YEARS AGO, WHEN I
STARTED THE UNIVERSITY OF
MARYLAND IS OUR INTEREST SO NOW
MY LAB WORKS ON INTERESTING
PARASITES INCLUDING LEISHMANIA
AND SO, AT THE END, IF YOU LOOK
AT THIS SCALE BAR, YOU CAN SEE
THE SCALE BAR WE KNOW MICE AND
FORMS ARE ALMOST THE SAME
BECAUSE FOR US, THE WARM IS AS
LARGE AS A MOUSE IN TERMS OF ITS
GENETIC POWER.
AND ITS IMPLICATIONS IN HUMAN
HEALTH AND DISEASE.
SO THE REASON I PUT THIS SLIDE
UP, IS BECAUSE PEOPLE OFTEN
CONFUSE HEME WITH GLOBINS.
HEMES ARE NOT JUST FOR GLOB
INNS, WHY DON'T YOU JUST PUT A
GFP TAG ON THE HEME, RIGHT.
SO THIS IS A MISNOMER BECAUSE
PEOPLE OFTEN CONFUSE HEME WITH
GLOBINS AND IT'S A PLANER RING
AND IT'S GOT TIE DEGREE PHOBIC
SITE CHAINS THAT MAKES THIS
MOLECULE HYDRO PHOBIC AND IN THE
BIOLOGICAL MEMBRANES AND CAUSE
TOXICITY BECAUSE OF THE IRON IN
THE MIDDLE THAT GENERATES
RADICALS AND THE RING ITSELF
CATALYZES IS IT'S CHARACTERIZED
AND OVER 400 CLASS OF GLOBINS
HAVE BEEN CRYSTALLIZED AND BIO
FISCAL AND BIOCHEMICAL BUT HERE
IS THE INTERESTING QUESTION, WE
DON'T KNOW HOW HEME GETS INTO
GLOBIN AFTER 70 YEARS.
THIS IS REALLY A PHENOMENALLY
OPEN QUESTION.
THESE ENORMOUS GAPS IN OUR
KNOWLEDGE.
HEMES ARE NOT FOR GLOB INS BUT
THEY'RE INVOLVED IN OTHER
INTERESTING BIOLOGICAL FUNCTIONS
SUCH AS SYNTHESIS OF NITRIC
OXIDE AND ENZYME AND AND THEY'RE
AND BEFORE 50 SIDE CHAIN ENZYMES
ARE INVOLVED IN STERILE
METABOLISM AND OX CASES IN ATP
SYNTHESIS AND REACTIONS AND THE
VOLUME PRODUCING PUSS AND NUTRI
FILLS AND THEY ARE INVOLVED IN
THE CONVERSION OF T3 TO T4 SO
HEMES PERFORM REALLY DIVERSE
BIOLOGICAL FUNCTIONS IN ALL OF
THESE CASES, THEY SIT AT THE
SIDE AND DO INTERESTING CHEMICAL
REACTIONS BUT A NEW CLAUSE OF
PROTEINS HAVE BEEN RECENTLY
DISCOVERED WHERE THEY'RE NOT
STATIC PROSTHETIC GROUPS DURING
CATALYTIC FUNCTIONS BUT THEY'RE
CO FACTORS INVOLVED IN
CONTROLLING BIOLOGICAL FUNCTIONS
AS SIGNALING MOLECULES AND SO
HEME BINDING TO TRANSCRIPTION
FACTOR ARE CONTROLS CIRCADIAN
CLOCK AND COUPLES WITH
METABOLISM AND BINDING ALLOWS
HOW MUCH MICRO RNA IS PRODUCED
AND TO THE POTASSIUM CHANNEL OR
THE ALLOWS YOU TO OPEN AND CLOSE
QUICKER AND SLOWER AND HEME
BINDING WITH A SYNTHESIS ALLOWS
HOW MUCH HYDROGEN TO GO PRODUCE
SO WITHIN THING IS CLEAR FROM
THIS SLIDE, THAT HEME IS THE
INTERSECTION OF FOUR GASES THAT
HAS PHYSIOLOGICAL GOALS.
SYNTHESIS AND HYDROGEN SULFIDE
AND IF YOU DEGRADE THIS MOLECULE
BY BREAKING THE METHANE BRIDGES
YOU OPEN UP THE RING, YOU
RELEASE IRON BUT YOU ALSO
PRODUCE CARBON MONOXIDE AND I'LL
COME BACK TO THAT LATER.
SO, THIS IS THE QUESTION THAT I
GOT INTERESTED 21 YEARS AGO WHEN
I WAS STILL A POSTDOC AT
WASHINGTON UNIVERSITY.
HEME IS MADE OF THE CELLS AND
IT'S MADE THROUGH A HEIDI
DEFINED BIOCHEMICAL PATHWAY
SHOWN HERE THAT IS SPATIALLY
SEPARATED BETWEEN THE
MITOCHONDRIA AND THE SITE OWE
SOL.
THESE ARE EIGHT INGVA INGVAR' AE
INSIMS THAT ARE CONSERVED ACROSS
ORGANISMS.
IN PLANTS, THE FIRST STEP IS
THROUGH A TRNA INTER MEDIATE.
THE IRON ENTERS IN THE LAST STEP
OF SYNTHESIS WHILE THE RING THAT
YOU SAW IN THE PROCEEDING SLIDE,
GETS INSERTED THROUGH A CAD A
LID I CAN REACTION BY AN ENZYME
AND THEN YOU MAKE THAT HEME THAT
YOU SAW IN THE PREVIOUS SLIDE.
IT'S MADE IN THE MITOCHONDRIAL
MATRIX AND THE QUESTION I GOT
REALLY INTERESTED IN, HOW DOES
THE HYDRO PHOBIC GET OUT OF THE
INTER MEMBRANE DO WE PUT INTO C
AND ALL THE HEME CONTAINING
PROTEINS THAT I GAVE YOU
EXAMPLES OF AND HOW DOES HEME
GET INTO GLOBE INNS, HOW DOES IT
GET INTO THE NUCLEUS TO PRESS
GOWNS AND HOW DOES HEME GET IN.
THE REASON WHY THIS QUESTION HAS
BEEN SO DIFFICULT TO ANSWER AND
IT'S BECAUSE OF HEME SYNTHESIS
ITSELF WHICH IS A HIGHLY
COMPLICATED AND REGULATED
PATHWAY.
IT'S REGULATED BY THE AMOUNT OF
IRON THAT IS AVAILABLE AND IT'S
REGULATED BY THE FLUX OF THE
STEPS BECAUSE THESE INTERMEDIATE
PRODUCTS ARE SUBSTRATES ARE
HIGHLY TOXIC SO YOU HAVE A TOXIC
SUBSTRATE AND A TOXIC PRODUCT
MADE BUT IT'S CRUCIAL FOR LIFE.
SO THEREFORE, HEME THIN A SIS IS
COUPLED WITH PROTEIN SYNTHESIS
REGULATED BY A FACTOR SUCH AS
IRON, HEME ITSELF AND GASES.
SO HOW'S TUESDAY ONE SPATIALLY
SEPARATE SYNTHESIS FROM
TRAFFICKING?
THAT WAS THE PROBLEM.
SO AS I GOT INTERESTED IN THE
QUESTION, MY COLLEAGUE JANE
UNCOVER A PROTEIN WHICH THEY
NAMED A PROTEIN THAT WAS TRANCE
MEMBRANE DOMAIN PROTEIN BINDS
HEME AND IS ABLE TO EXPORT HEME
THE WAY SHE UNCOVER IT WAS
SHOWING THAT CATS INFECTED WITH
THE DELINEFELINE LEUKEMIA IS WHE
VIRUS INFECTS IT BINDS TO THIS
PROTEIN, INTERNALIZES IT AND
DEGRADES IT AND CAUSES APOPTOSIS
OF RUN CELLS BECAUSE THEY'RE
UNABLE TO EXPORT HEME OUT.
WHILE THIS WAS A DECADES-LONG
INQUIRY INTO WHY, HOW FELINE
LEUKEMIA CAUSES RED CELL A
POLICE STATION I AND WANTED TO
SEPARATE SYNTHESIS FROM
TRAFFICKING.
SO WHEN I WAS STILL A POSTDOC AT
WASHU, I WAS REALLY CONSIDERING
HOW DO I GO ABOUT ANSWERING THIS
QUESTION.
SO, HERE ARE THE SEVEN STEPS OF
HEME SYNTHESIS.
I HAVE NOT PUT THE FIRST ENZYME
BECAUSE THEY'RE DIFFERENT
BETWEEN PLANTS AND NON PLANT
ORGANISMS.
AS YOU CAN SEE FROM THE BLAST
MALL I COULD PUT THE SAME
SYNTHETIC PATHWAY ENZYMES.
IF YOU LOOK AT THE GEE MOM WE
COULD NOT FIND ANY HOMO.
THIS WAS REALLY INTERESTING
BECAUSE WHEN BOB WATERSON
PUBLISHED THIS IN 1997, NONE OF
THESE GENES WERE IDENTIFIED AS
POSSIBLE AND THEY MISSED A PART
OF CHROMOSOME AND IT'S ALSO
POSSIBLE THAT C ELEGANS DOESN'T
CARE FOR IT SAYS SOME ORGANISMS
AND MICRO ORGANISMS.
THE OTHER POSSIBILITY IS C.
ELEGANS MAKES IT THROUGH A
PATHWAY WHICH WOULD BE VERY
INTERESTING AND THE FOURTH
POSSIBILITY WAS THAT C. ELEGANS
DOES NOT MAKE HEME BUT ACQUIRES
IT.
THIS IS WHERE I TOOK A
THREE-WEEK COURSE AT COLD SPRING
HARBOUR BECAUSE I THOUGHT MAYBE
IT CAN ESPECIALLY SEPARATE
SYNTHESIS FROM TRAFFICKING.
AFTER TAKING THE COURSE I
REALISED THAT C. ELEGANS IS
BACTERIA.
E. COLI IN THE LAB AND E. COLI
HAS THE PATHWAY SAME WAY THAT
YOU AND I DO.
SO HOW CAN CAN ONE GO WORMS BY
STILL CONTROLLING THE LEVELS OF
HEME?
SO, I WAS ABLE TO SEND MY
AMERICAN POSTDOC TO THE U.S.
MILITARY BASE AT FORT DETRICK
BECAUSE THE U.S. ARMY WAS
INTERESTED IN USING C. ELEGANS
AND THEY HAD LIQUID CULTURE THAT
THEY NAMED CH R AND SHE CAME TO
THE LAB AND I DO NOT GE COULD NN
THE MILITARY BASE AND I SAID
WHAT IS IN THE LIQUID SYNTHETIC
MEDIUM AND IN SIGHT OWE CHROME
C.
IF YOU TAKE OUTSIDE FROM THIS
LIQUID CULTURE THE WORMS DON'T
GROW AND IF YOU START ADDING
PURE HEMEN, THEY GROW WELL AND
THERE'S A 20 MICRO MOLAR AND AS
YOU INCREASE THE CONCENTRATION
OF THE HEME, WORM STOPS GROWING
AND EVENTUALLY IT BECOMES REALLY
TOX SICK THIS WAS A YEAR AFTER
IT STARTED MY LAB AT MARYLAND
STILL TRYING TO UNCOVER WHERE
THE C. ELEGANS MAKE HEME OR
ACQUIRES HEME EX.
WE CAN DO SOME INTERESTING
EXPERIMENTS INCLUDING WITH HEME
TO SHOW IN DEED C. ELEGANS DOES
NOT MAKE HEME BUT GET IT EX --
THE LASTEN TIME AND NONE
DETECTED AND FOR ELEGANS AND
SINCE WE'RE IN CLOSE PROXIMITY
TO THE USDA I GOT TO THE HEMA
POLL GEE LAB AND LOOKED AT OTHER
FREE-LIVING AND WE WENT TO THE
ANIMAL HEMATOLOGY LABORATORY AND
THIS IS A HUGE EDGE DOES TREE,
PARASITIC ALSO HAULED HELMINTH
SO WE HAVE YOUR FRED WORK, HOOK
WORM, AND ROUND WORM AND IN
FACTS HUMAN LIVE STOCKS AND
PLANTS.
THIS IS IS PRE GENOME ERA AND WE
KNOW C. ELEGANS ARE ALL HEME AND
THEY HAVE LOST ALL A GOMES
INFORM SYNTHESIS SO IF YOU HAVE
BEEN TO SAY WORM LAB, I'M
TELLING YOU IT'S A POWERFUL
MODEL SYSTEM FOR US
SPECIFICALLY.
SMALL SIZE, A LOT SMALLER THAN
MICE YOU CAN DO SOME POWERFUL
GENETICS OF TRANCE LOOSE ENTER
FROM AN EGG LAYING ADULT AND
SHORT LIFE CYCLE, THREE AND A
HALF DAYS, EACH HERMAPHRODITE
LAYS BETWEEN 300 TO 500 PRO GENI
CAVITT AND AND EACH CELL HAS BEN
MAPPED.
THE ENTIRE ANIMAL HAS BEEN
SECTIONS FROM THE MOUTH TO THE
SAIL AT EM LEVEL, RIGHT.
AND FOR US, THE MOST POWERFUL
THING FOR US, THAT WE CAN GROW
THEM IN A LARGE-SCALE TO DO
INTERESTING METABOLISM AND BY
OWE CHEMICAL STUDIES BECAUSE IT
IS A NATURAL MUTANT FOR HEME
SYNTHESIS SO AS GONE OWE GENOTYO
NOT MAKE HEME BUT ACQUIRE HEME
FROM THE ENVIRONMENT.
SO SINCE C. ELEGANS IS AN
ANIMAL, THE HERMAPHRODITE HAS 20
BEAUTIFUL POLARIZING CELLS
THROUGH WHICH IT EATS AND IT HAS
ABSORBED AND STORAGE FUNCTIONS
AND SO HERE IS A SINGLE CARTOON
OF A POLARIZING INTESTINAL CELL
AND THIS IS THE LUMIN OF THE
INTESTINE AND SINCE IT ACTS IT
TAKES UP HEME FROM ITS FOOD AND
DIRECTLY INCORPORATES IT AS WE
HAVE SHOWN USING RADIO LABEL
TRACER EXPERIMENTS INTO HEME
PROTEINS AND WE HAVE 33
DIFFERENT GLOBE INNS AND THEY
BLIND HEME AND OVER 84 PEOPLE
AND SO WE HAVE COMPLETE CONTROL
OF THE FLUX OF HEME ALL WITHIN
THE CONTEXT OF A TRANSPARENT
ANIMAL APPROXIMATE MORE
IMPORTANTLY, NOT ONLY AND
INCORPORATING TO PROTEINS BUT WE
CAN TAKE IT FROM THE INTESTINE
AND TRANSPORT IT ACROSS THE
INTESTINE TO ALL THE OTHER
TISSUE, MUSCLE, NEURONS, AND THE
PROGENY.
SO HERE IS THE FIRST PIECE OF
DATA, IF YOU GROW WORMS UNDER
THE LOWEST HEME CONCENTRATION
WHICH IS 1.5 MICRO POLAR HEME
AND THEY TAKE NINE DAYS TO GO
HERE THEY'RE OPTIMAL AND THEY
ACQUIRE THREE DAYS TO FREE AND
AND YOU GIVE THEM A ANALOGUE
CALLED SINC HOMEOSTASIS AND YOU
CAN CAN TAKE THE IRON AND PUT IN
OTHER MEDALS SO IT'S A AND YOU
CAN CAN KNEW HOUSE IT AND WE
HAVE IT WITHIN THE THEY DON'T
TAKE IT UP AS WELL AND EVEN
THOUGH HEME IS ESSENTIAL AND
THAT WOULD MEAN THAT THEY'RE
UPTICK SYSTEM NEEDS TO BE
REGULATED.
YOU CAN QUANTIFY THIS
FLUORESCENS INTENSITY HERE SO
THE GROWTH CURVE I SHOWED YOU
DIFFERENT IN CONCENTRATIONS AND
HERE IS THE FLUORESCENT HEME
ANALOGUE SO WORMS GROW AT LOW
HEME CONDITIONS TAKE UP HEME
EFFECTIVELY AND WARMS GROW AT
HIGH FEMALE CONCENTRATIONS AND
SWITCH IT OFF.
IT'S REGULATED AND SO THIS
ALLOWED US TO USE THREE
DIFFERENT APPROACHES.
UNBIASEDLY, AGAIN ET TICKS,
GENETICS, GENETICS, WITH THE
ENTIRE GENOME USING EMS AND
REVERSE GENETICS KNOCKING DOWN
ALL 19,000 GENES ONE AT A TIME
AND ENDING THE TRANSCRIPT TOPICS
IDENTIFYING HEME RESPONSIVE
GOWNS.
HERE IS WHAT WE HAVE DISCOVERED
USING, THIS IS WHERE I USE THE
TERM THE AWESOME POWER OF C
ELEGANS AGAIN GENETICS.
THE FIRST RESPONSIVE GOWNS ARE
FOLLOW THREE AND NOW FOUR-LETTER
CODES.
SO THESE ARE HEME IMPORTERS
SINCE C. ELEGANS DOESN'T MAKE
HEME IT'S DUPLICATED AND IT
LOCALIZES TO THE COMPARTMENT
FOUR, FIVE AND SIX AND A SMALL
PEP SIDE THE MOTHER SECRETS THAT
TAKES THE HEME AND DELIVERING IT
TO THE EMBRYO AND LOCALIZED IN
THE HYPER DERMIS ALLOWS THE
HYPER DERMIS TO UTILIZE HEME
EFFECTIVELY AND HEME HE CAN
PORTER WHICH LOCALIZES TO THE
SURFACE EXPORT HEME TO ALL THE
OTHER TISSUES IN ITS ABSENCE AND
THE MOTHER AS WELL AS THE
EMBRYOS ARE DEAD AND THIS IS
UNDER LOW HEME CONDITIONS
INTERNALIZES IN THE SENSORY
NEURONS AND THE FENCE REAND HOME
A LOG CALLED DOUBLE 1 WHICH
TURNS ON THIS MAD TRANSCRIPTION
FACTORS AND REMESSES THIS GENE
SO THIS IS THE ACCELERATOR AND
THIS IS THE BREAK WHICH A LOUSE
THE ENTIRE ORGANISM TO RESPONDS
TO DIETARY HEME BECAUSE ALL OF
IT IS DEPENDING ON THE HEME
COMING FROM THE FOOD AND THROUGH
THE INTESTINE.
SO THE ULTIMATE GOAL FOR US
REMAINS CAN CAN WE EXPLOIT C.
ELEGANS TO IDENTIFY PATHWAYS AND
VERTEBRAES AND CAN WE EXPLOIT
DIFFERENCES BETWEEN THE HUMAN
ACQUISITION PATHWAYS TO THE
PARADISE BECAUSE AS I MENTIONED
BEFORE, MOST ARE HELMINTH TO NOT
HAVE THE CAPACITY TO TRANSPORT.
THIS I'M GOING TO DISCUSS ABOUT
ONE TRANSPORTER TODAY WHICH WE
NAMED HRG1 OR NOW IT'S CALLED
HRG41.
IT WAS PROTEINS AND GENES THAT
ARE HIGHLY EXPRESSED UNDER LOW
HEME CONDITIONS.
THEY'RE REGULATED TRANSCRIPTION
LOW AS WELL AS TRANSLATIONALLY
AND THEY ARE PARALLEL
TRANSPORTERS AND C. ELEGANS HAS
FOUR OF THEM AND THESE TWO ARE
HIGHLY RESPONSE SHOWN HERE AND
FIVE OR SEX OR REGULATED AT A
POST TRANSCRIPTION LEVEL, WE
HAVE ONLY ONE OF THESE
TRANSPORTERS SO I'M GOING TO
DISCUSS THROUGH THIS MANY
DIFFERENT ORGANISMS AND WITH THE
ROLE OF THESE FIRST CLASS OF
HEME IMPORTERS LOOK LIKE.
SO THESE ARE FOUR TRANCE
MEMBRANE DOMAIN PROTEINS WHICH
IS NOT WHAT I WOULD HAVE
PREDICTED.
THEY ARE 570 ORGANICS AND
MOLECULES AND I WOULD HAVE
THOUGHT THAT THESE TRANCE
MEMBRANE DOMAIN PROTEINS HAVE
EIGHT TO 12 PROTEINS SO I CAN
SAY AT THIS POINT IS THESE ARE
TRIMER IF NOT A AT THE TIME TREE
OK AND THIS IS H RG FOUR AND
LACKS GREN AND BLUE RESIDUE AND
FOR THOSE PEOPLE THAT ARE
INTERESTED IN MEMBER AND
TRAFFICKING, THIS GREEN RESIDUE
ARE SORTING MOR TIVE AND BASED
MOW TEASE AND FOR RETREATS OF
THE PROTEIN FROM THE Mrs. MA
WOMEN RAIN TAKING PROTEINS TO
THE LYSOZOME FOR LAX THOSE AND
IT HAS BEEN REPLACED BY A SCENE
AND REFERS BINDING AND REACHING
WITH OXIDIZED STEAM THE HUMAN
AND BUT IF YOU LOOK AT IT IT'S
PROTEINS BUT THOSE CRITICAL
RESIDUES THAT HAVE SHADED IN
RED, RIGHT HERE, THEY'RE HEALEY
CONSERVED IN THE HUMAN PROTEINS
AND IT YOU KNEW TAY THOSE AND
THE TRANSPORTER IS DEAD AND IT'S
UNABLE TO TRANSPORT AND SO WHAT
DO THESE GUYS LOOK LIKE IN VIVO?
HERE WE SEE C. ELEGANS THESE ARE
THE INTESTINAL FOR ALLIESED
CELLS AND OPPOSING EACH OTHER
AND THIS IS THE DARK LUMIN
BETWEEN AND IT LOCALIZES AND I
CAN CAN SEE FOR SURE, THIS IS
THE FIRST HEME ANALOGUE AND
THESE ARE THE OREGONELS AND THE
COMPLETELY SURROUND THESE LROs
AND IT'S LAX THE GREEN AND BLUE
RESIDUE AND THEY LOCALIZE TO THE
PLASMA MEMBRANE AND THIS DARK
SIDE IS THE LIEU MINUTE, YOU CAN
CAN CHANGE THE LOCALIZATION BY
FLIP-FLOPPING SO IT'S
LOCALIZATION IS DICTATED BY C
AND HERE IS THE HUMANIZED WORM.
WE HAVE DELETED ALL FOUR
TRANSPORTERS AND PUT IN THE
HUMAN GENE TO THE WORM AND IT
FULLY RESCUES THE C. ELEGANS
QUADRUPLE MUTANT AND IT
LOCALIZES TO THE PLASMA MEMBER
BAINMEMBRANESO IT'S TAKEN OVER N
OF BOTH HRG1 AS WELL AS FOUR.
SO THE QUESTION THAT WE ALWAYS
GOT WHEN WE SUBMIT A PAPER TO A
JOURNAL, IS THIS -- DO MA MALE
YAN CELLS UTILIZE HEME BECAUSE
ALL OF THE WORK WITH THE C.
ELEGANS STUFF TELLS US YES C IS
A HEME OK TROVE AND IT
TRANSPORTS HEME.
SO THERE'S ABSOLUTELY NO
EVIDENCE THAT HEME HAS A SINGLE
MOLECULE MOVED DIRECTLY FROM
CELL TO CELL IN MAMMALS AND
LIKELY ALL VERTEBRAES.
AFTER HEME MADE A MAMMAL AN CELL
IT REMAINS UNTIL THE PROTEINS
CAT ABOLISED.
I USED THESE REVIEWS TO PIVOT
MYSELF AND REFOCUS.
SO, THE WAY TO ADDRESS THIS
QUESTION WAS TO SEE DO MA MALE
YAN CELLS AND IT'S BEEN LOAN IN
THE 1970s THAT PATIENTS AND THEY
HAVE SYNTHESIS IS THESE
INDIVIDUALS CAN BE TREATED WITH
HEME.
THAT WOULD MEAN THAT SALTS COULD
TAKE UP HEME IF GIVEN EX ON
OBVIOUSLY SO WE ADDRESS THE THE
QUESTION IN A DIFFERENT WAY.
WE TOOK TWO PEROXIDASE AND
THEY'RE BOTH PLANT PROTEINS AND
THEY BIND HEME WITH THE
RELATIVELY DESEPTEMBER AFFINITY
OF 270 TO 350 NANA MOLAR AND
THEN WE TOOK THOSE PEROXIDASE
AND LOCALIZED IT TO OREGON HEELD
THE Mr PLASMA MEMBRANE AND THE
NUCLEUS AND WE ASKED THIS
QUESTION, DO MAMMALIAN SELLS
USE.
WHAT WE CAN DO IS TAKE OFF ANY
CONTAMINATING HEME AND THERE IS
SOME IN THE SERUM IN CULTURAL
CELLS SO WE CAN HEME DEPLETE
THEM AND SWITCH OFF THE PATHWAY
USING A CHEMICAL CALLED AS OWE
TEEN WHICH BLOCKS THE SECOND
STEP OF THE PATHWAY AND BECAUSE
IT'S HRP IT CAN DO AND USING
DIAMOND OWE BENCE DEAN YOU CAN
DO PLATE READERS OR YOU CAN TO
MYRIOSCOPY SO WE CAN CANNOT
BADLY DETECTIVE TEE IN ALL OF
THESE SIX COMPARTMENTS AND YOU
TURN ON THE FAUCET FROM THE
OUTSIDE SO YOU BRING IN FROM THE
OUTSIDE AND YOU RETURN ONE TO
FOUR MICRO MOLAR IS SUFFICIENT
TO RESTORE HEME LEVELS IN ALL OF
THOSE COMPARTMENTS SO THESE ARE
A RESULT SAY THAT MAMMALIAN
CELLS ARE FLEXIBLE AND THEY CAN
MAKE HEME BUT IF IT SWITCHES OFF
THEY REQUIRE HEME FROM THE
OUTSIDE THEY CAN TAKE IT UP.
SO LET'S GO BACK TO THIS
QUESTION.
TO MAMMALIAN CELLS REQUIRE A
HEME TRANSPORTER?
SO, WE MADE ANTIBODIES AGAINST
THE HUMAN HOM OWE LOG OF C
ELEGANS HRG1 AND THIS IS HIGHLY
ENRICHED MACROPHAGES.
AND THEY'RE IN THE SYSTEM AND
THE RES MACROPHAGES LAY A REALLY
CRITICAL ROLE IN IRON METABOLISM
AND WE HAVE 25 TRILLION RED
BLOOD CELLS AND EACH RED BLOOD
CELL CONTAINS ABOUT A BILLION
HEME MOLECULES AND EVERY SECOND
JUST LIKE THIS, WE'RE RECYCLING
FIVE MILLION RED BLOOD CELLS
BECAUSE THE LIFESPAN OF A RED
CELL IS 120 DAYS IN HUMANS AND
IN MICE BETWEEN 40 TO 60 DAYS.
SO AS THEY UNDERGO THEIR OUTSIDE
MEMBRANE SLIGHTLY DIFFERS AND
THESE ARE RECOGNIZED BY THE
RECEPTOR IN THEIR SYSTEM
MACROPHAGES.
AND THE PHAGO CYTO SIS ARE DYING
RED BLUE CELLS AND IT MATURES
INTO THE COMPARTMENT AND THE RED
CELLS ARE DEGRADED AND THEY'RE
RELEASED THE HEME.
THIS HEME IS THEN IMPORTED INTO
THE SITE OWE SOL AND IT DOGMA IS
THAT THIS HEME IS THEN
EXCLUSIVELY DEGRADED BY HEME
OXYGENASIS TO OPEN UP THE RING
BY BREAKING UP ONE OF THE
BRIDGES AND RELEASING THE IRON,
CARBON MONOXIDE AND MAKES THAT
LINEAR RING NOW INTO IT GETS
CONVERTED TO BILLY RUBEN IN
EXCRETED OUT.
THE IRON THAT IS RELEASED THEN
IS EXPORTED OUT BY A PICKED UP
BY THE TRANSPARENT PROTEIN IN
THE CIRCULATION AND TAKEN TO THE
BONE MARROW AND PICKED UP BY THE
RECEPTORS AND THE BONE MARROW TO
MAKE NEW RED BLOOD CELLS AND THE
EXCESS GETS STORED SO HERE IS
THE OPEN QUESTION, RIGHT.
EVERY SECOND, WE DEGRADE FIVE
QUADRILLION MOLECULES TO MAKE
FIVE MOLECULES BACK IN THE BONE
MARROW FOR NEW RED CELL
PRODUCTION.
THAT IS THE CURRENT THINKING.
AND I CAN BE HAPPY TO ANSWER
QUESTIONS FOR THAT.
SO THE QUESTION WE ASKED WAS,
GIVEN THE FACT THAT IT'S HIGHLY
EXPRESSED IN MACROPHAGES IT
LOCALIZES TO THE HIGH SO SOME IS
IT POSSIBLE IT'S THE MACROPHAGES
TRANSPORTER THAT PUTS IT
UPSTREAM GENETICALLY AND CELL
BIOLOGY AND TO DO THIS, WE
ISOLATED BON MARROW MACROPHAGES,
AND WE FOUND OUT THAT IT I AM I
CAN LOW YES IN BONE MARROW
MACROPHAGES IT LOCALIZES TO THE
LATE END OWE AND IT WILL GO DECK
RAID THE COMPARTMENTS AND IT
REALIZES IT'S BEEN TRICKED AND
GOES BACK TO THE LATE
COMPARTMENT BUT IF YOU GIVE THEM
OX SA DIESED RED BLOOD CELLS,
THOSE HRG1 WILL GET ENRICHED AND
SHOWN HERE AND THIS
SYSTEMATICALLY CONTINUOUSLY IS
BEING RECRUITED FROM THE GUY
SHOWN HERE.
SO IS HRB1 UPSTREAM IS FERRATIN
SO IF YOU ARE NOT, WE GET ABOUT
80 TO 90% KNOCK TOWN AND NO IF
YOU FED THEM RED BLOOD CELLS BY
THE PROCESS, YOU CAN CAN SEE
THAT DURING HEME OXYGEN IS
IMPORTANT BUT IF YOU KNOCKDOWN
HRG1, AWFUL THOSE DOWNSTREAM
MARKERS ARE REDUCED SO THESE
RESULTS TELL YOU THAT YES,
BIOLOGICALLY HRG1 IS UPSTREAM
FROM ALL OF THROW MAJOR
CONTRIBUTING.
SO WE KNOCKED ON HRG1 IN MICE
AND IT WAS DID NOT RIGHT HERE
WITH MY FRIEND DAVID BOWDINE.
THE INITIAL HYPOTHESIS WAS
SIMPLE.
A KNOCKOUT MOUSE WILL BE
EMBRYONIC.
THESE MICE LOOK
INDISTINGUISHABLE FROM Y TYPE
MICE WHEN YOU LOOK FROM THE
OUTSIDE.
UNTIL YOU OPEN UP AND DISSECT
THE MOUSE AND OPEN UP THE RIB
KINGS ARE TARKANIAN AND YOU LOOK
AT THE SPLEEN, THE SPLEEN IS
DARK AND THE LIVER IS
DISCOLORED, AND THIS IS WHAT A
SINGLE FEMALE LOOKS LIKE FOR A
KNOCK OUT SO IT'S REALLY DARK
BUT IT'S NOT THE BONE ITSELF
BECAUSE IF WE FLUSH OUT THE
MARROW, THESE ARE A SINGLE CELL
WITH THIS DARK BLACK PIGMENT
WITHIN THE CELL AND HERE IS THE
MARROW.
SO THERE'S SOMETHING REALLY
UNUSUAL ABOUT THESE MICE.
IF YOU LOOK AT THE THREE TISSUE
THE SPLEEN, LIVER AND BON
MARROW.
THEY'RE THE REALLY DARK PIGMENT
THAT APPEARS TO HAVE
PRECIPITATED WITHIN THESE CELLS
AND THESE CELLS ARE MACROPHAGES.
WE CAN GRIND THEM UP AND HAVE
THE DARK PIGMENT AND THE ONLY
PIGMENT KNOWN TO CONSIDER IN
LIVING BIOLOGICAL SYSTEMS THAT
WE KNEW OF WAS HEMA SEW AN AND
UNTIL THEN IT WAS DISCOVERED TO
BE MADE BY PLAS MODE YUM
PARASITES.
AND SEE GUESS TERES AS CRYSTALS
INTO ITS LIE SO SOME AND THE
BUFFER AND TO THIS PALLET WHICH
WAS INSOLUBLE AND A COMPLETE IT
IS DISSOLVED AND GIVES YOU THE
BEAUTIFUL HEME SPECIFIC TRUST
AND WE CAN TAKE THIS BALANCE AT
AND DO SOMETHING DEFRACTION AND
WE GOT BACK THE STRUCTURE AND IS
IDENTICAL TO PLAS MODE YAN.
SO THESE RESULTS TOLD US THAT
YES, THE MOUSE APPEARS TO BE
NORMAL BECAUSE IT'S TAKING UP
THE PEOPLE IN THESE MACROPHAGES
AND BRINGING THEM TO THESE
CRYSTALS AND THIS IS WHY THE
PLAS MODE YAN CRYSTAL LOOKS LIKE
AND IN SCANNING, EM, HERE IS
WHAT THE LIVER AND BONE MARROW
LOOKS LIKE AND EVEN THOUGH
THERE'S SURFACE LOOKS SLIGHTLY
DIFFERENT THAN PLAS MODE YUM,
THEY'RE THE ULTRASTRUCTURE LEVEL
AND THIS IS THE BEAUTIFUL HRG1,
POSITIVE COMPARTMENT AND A HRG1
NULL MOUSE AND THEY'RE PSEUDO
COLOURED FROM BLACK TO RED AND
THEY'RE IN THIS SURROUNDED BY
THE LYSOZOME MEMBRANE AND
THEY'RE LARGER IN THE KNOCK OUT
AND SO THESE MACROPHAGES ARE
ACCUMULATING HEME IN THE FORM OF
HEMA SEW ININ THIS LYSOZOMAL
COMPARTMENT SO HOW AND WHY DO
THESE MICE MAKE HEMEAZOIN AND
WHAT IS THE BASIS FOR TOLERATING
THE HEME.
TO ADDRESS THAT WE TOOK A DONOR
MOUSE, A WILD LIFE MOUSE,
INJECTED WITH THE COMPOUND AND
IT CAUSES GIVING THEM AND 59 AND
AFTER THREE DAYS WE CAN
SACRIFICE THIS MOUSE AND
MAJORITY OF THIS RADIO LABEL
GETTING THE RED CELLS AND THE
HEMOGLOBIN WILL GET LABELLED BY
IN59 SO IT IS 89% GETS
INCORPORATED INTO HEME AND WE
CAN TAKE THIS LABELLED RED CELLS
AND DAMAGE THEM AND GIVE THEM TO
RECIPIENT MICE IN THE WILD TYPE
AND KNOCK OUT AND SACRIFICE THE
MOUSE LATER TO LOOK AT THE
CONNETICS OF CLEARANCE OF THESE
RED CELLS AND HERE IS AN
EXAMPLE.
JUST LOOKING AT THE SPLEEN.
WILD TYPE MICE CLEAR MOST OF
THESE LABELLED RED CELLS AND
THIS IS ALL THAT IS REMAINING
AFTER 96 HOURS AND HERE IS THE
KNOCK OUT MICE AND DUNCE OF
RADIO LABELLED IRON AND WE CAN
DISTINGUISH HEME FROM NON
EASTIRON ANDYOU CAN SEE THERE'SA
SIGNIFICANT AMOUNT OF HEME STUCK
IN THE ORGANIC FACE IN THE KNOCK
OUT.
BUT THIS PLUS NON HEME IRON DOES
NOT EQUATE TO THAT AND WE'RE
LOSING SOMEWHERE SO IF YOU ARE
NOW LOOKING AT THE PALLET, THE
MAJORITY OF THE IRON 59 COUNTS
ARE IN THE PALLETE, YOU CAN CAN
TAKE THAT HEMA SEW INPEL AT AND
USE TH DISSOLVING BUFFER AND ITS
WHERE IT'S GOING AND HERE IT IS,
THE MAJORITY OF THE IRON 59 IS
GOING INTO HEMA ZONE HEAT.
SO WHY ARE THESE MICE SURVIVING
IF THEY'RE UNABLE TO RECYCLE THE
HEME IRON.
BECAUSE A NORMAL IRON IS FED WAY
TOO MUCH IRON IN THEIR CHILD AND
A TYPICAL MOUSE NEEDS ABOUT 20
BITES HER MILLION OF IRON AND
STANDARD HAS 380 PARTS PER
MILLION SO IF YOU TAKE THESE
WILD TYPE AND KNOCK OUT MICE AND
FEED THEM LOW IRON DIET WHICH
TWO PARTS PER MILLION, BOTH THE
WILD TYPE AND KNOCK OUT MOUSE
DROPS ABOUT 45 AND DONE ALL THE
WAY TO 20 FOR THE FIRST FIVE
WEEKS HERE IS YOUR CONTROL.
THEY REMAIN NOT SIGNIFICANTLY
DIFFERENT UNTIL AFTER FIVE
WEEKS, AFTER FIVE WEEKS, THEY
REACHED A NEW STUDY STATE LEVEL
AND WHERE THEY'RE SOLELY RELYING
UPON RECYCLED IRON AND AS THE
KNOCK OUT BECAUSE IT'S UNABLE TO
RECYCLE IRON CONTINUOUSLY
ACCUMULATES HEMA ZONE AND
EVENTUALLY THEY SUCCUMB BECAUSE
OF SEVERE ANAEMIA SO THERE'S
MASSIVE CHANGES IN GENE
EXPRESSION BETWEEN THE WILD TYPE
AND KNOCK OUT MOUSE IF YOU LOOK
AT THE GENE EXPRESSION PROFILE
AT FIVE WEEKS.
SO THE MODEL FOR HOW DOES H RG1
KNOCK OUT MOUSE SURVIVES AND AS
I SAID BEFORE, BODY IRON IS A
COMPOSITE OF THE AMOUNT OF
EASTERN COMING FROM FOOD AND
VERSUS THE AMOUNT OF IRON THAT
IS BEING RECYCLED AND IF THROW
MAJOR ORGANS SPLEEN, LIVER, AND
BONE MARROW AND BONE MARROW
PRODUCES NEW RED CELLS IN THE
ABSENCE OF HRG1 THERE'S NO
RECYCLING GOING ON FROM THE
SPLEEN, LIVER OR MARROW THE
SPLEEN ACCUMULATES AND LIVER
STARTS MAKING HEMA SEW INAND THE
BONE MARROW STARTS MAKING AND
EVENTUALLY YOU WILL END UP IN
SOMETHING CALLED INEFFECTIVE SO
THE BONE MARROW SAYS SPLEEN,
COULD YOU GIVE ME A HAND?
THE SPLEEN TRIES TO EXPANDS BUT
IT CAN'T UNDERGO STRESS TO HELP
MAINTAIN NORMAL AND EVENTUALLY,
THESE MICE START DEVELOPING
LARGEMENTS AND THEIR ORGANS ARE
MESSED AND THEY'RE LARGE LIPID
DROPLETS BUT THEN TISSUE AND
THESE MICE CONTINUE TO SURVIVE
EVEN THOUGH THEY'RE UNABLE TO
RECYCLE BECAUSE THEY'RE GETTING
A LOT OF THEIR IRON TO BE ABLE
TO MAKE NEW RED BLOOD CELLS IF
YOU REMOVE THE IRON SOURCE AS WE
DID UNDER LOW IRON CONDITIONS,
THESE MICE DIE.
I'M GOING TO DISCUSS HOW DO
PARASITES ACQUIRE HEME.
AS I MENTIONED BEFORE, IF IT'S
MORE OR LESS HIGHLY CONSERVED ON
THIS PLANET AND WE HAVE SHOWN
THAT PARASITIC C. ELEGANS LACKS
THE ABLE TO SIN THAT SIGHS BUT
HIGHJACKS HEME FROM THE OUTSIDE
AND SO WHAT DOES THAT MEAN IN
TERMS OF AN EVOLUTIONARY
PERSPECTIVE?
WOULD THAT MEAN THAT THE
ANCESTORIAL WORM OR THE INAS
TREAL ORGANISM WAS A PARASITE
THAT WAS LIVING IN A HEME-RICH
ENVIRONMENT THAT UP IT SAID WHY
TO I NEED TO MAKE IT WHEN I CAN
EAT IT?
OR IS IT POSSIBLE THAT IT WAS
LIVING IN AN RELATIONSHIP SUCH
THAT IT WAS PROGRESS HEME IN
EXCHANGE FOR KNEW TREMENDOUS
ENTERS FROM THE HOST, WHILE BOTH
OF THIS SCENARIOS COULD BE TRUE,
HOOK WORMS LIVE IN THE INTESTINE
AND GEORGES ON HUGE AMOUNTS OF
BLOOD AND AN BACTERIA THAT LIVES
WITHIN MALAI AND IF YOU KNOCK
OUT THE PATHWAY AND IT DIES AND
NONE OF THESE ORGANISMS ARE
GENETICALLY TRACTABLE AND TO BE
ABLE TO GET TO A GENETICALLY
TRACTABLE ORGANISM WE STARTED
WORKING WITH THE PARASITE VISH
MANIA AND IT LACKS THE APPARATUS
FOR HEME SYNTHESIS AND THE MOST
INTERESTING THING FOR US IT'S
TRANSMITTED BY FLIES SO IT HAS A
STAGE AND WHEN IT'S INJECTED
INTO THE HOST, THEY INVADE AND
THE MACK MACROPHAGES IN THE CASE
OF LEISHMANIA AND THEY LIVE
INSIDE THE SEEM OF THE SAME
MACROPHAGES RECYCLING ALL OF THE
HEME THE IRON AND THESE CONTINUE
TO REPLICATE INSIDE THE
MACROPHAGES UNTIL THEY CAUSE THE
MICE OF THE HOST RED CELLS NEW
RELEASED AND THEY INFECT
NEIGHBORING CELLS.
SO THE BEAUTY OF LEISHMANIA IS
THAT THEY ARE GENETICALLY
ATTRACTABLE AND YOU CAN KNOCK
OUT GENES, YOU CAN CAN KNOCK IN
GOWNS, YOU CAN RESOLVE THEM BY
GREG THEM IN LIQUID CULTURE AND
YOU CAN INFECT MACROPHAGES IN
CELL CULTURE TO LOOK AT HOST
PARA SIDE INTERACTIONS AND YOU
CAN DO IN VIVO EXPERIMENTS WITH
GENETICALLY ALTERED MICE OR
HAMSTERS.
SO, BY INTERROGATE SOME
INTERESTING BIO FORMAT TICKS WE
IDENTIFIED THE FIRST PARASITE SO
WE JUST NAMED HIM LEISHMANIA
HRG1 AND LAUCH MANIA IS A DIP
LLOYD SO IF YOU MAKE A SINGLE
KNOCK OUT AND MAMMALIANS AND THE
AMOUNT OF HEME TROOPS BY HALF
YOU CANNOT MAKE A DOUBLE KNOCK
OUT BECAUSE IT IS LETHAL AND YOU
CAN PUT IN A TRANS GENE AND YOU
CAN RESTORE HEME LEVELS ALMOST
AS GOOD AS WILD TYPE.
HERE IS A SINGLE KNOCK OUT FOR
LHR1 AND IT'S UNABLE TO FORM
LESIONS IN MICE COM PARED TO
WILD TYPE MICE AND GENETICALLY
RESCUED.
SO REGULATES DIFFERENTLY AND IN
THE ABSENCE OF HEME, LHR1 IS UP
REGULATED AND THIS PAR SIGHT CAN
CANNOT MAKE HEME.
IT REQUIRES HEME TO SURVIVE SO
HAS TO HIJACK HEME SO THE
PROTEIN IS TURNED ON IN THE
ABSENCE OF HEME AND AS HEME
CONCENTRATION GOES UP AND DIALS
IT DOWN AND UNLIKE THAT, HUMAN
HRG1 IS TURNED ON WHEN THERE'S
HEME AS YOU WOULD EXPECT WHEN
IT'S ACQUIRED TO UNDERGO SO THE
DYNAMICS AND REGULATION OF
MACROPHAGE HRG1 AND HOST HRG1
ARE QUITE DIFFERENT.
SO HERE IT IS, THIS IS HOST HRGI
ON THE MACROPHAGES AND THEY ARE
INFECTED BY LAUCH MANIA AND IT'S
A OXY OPEN AND IT REQUIRES HEME
FOR ITS OWN SURVIVAL AND IT HAS
A DIFFERENT VARIATION OF THE
SAME FOUR TRANS MEMBRANE DOMAIN
PROTEIN, IT'S TRYING TO HIJACK
THE SAME SUBSTRATE OR NUTRIENT
TO BE ABLE REPLICATE AND DIVIDE.
THE INTERESTING THING, IS THAT
LHR1 AND HRG1 ARE ONLY 13%
IDENTICAL.
WHICH HAS IMPLICATIONS FOR THE
NEXT SLIDE.
SO THE QUESTION WE ARE ASKING IS
THAT DOES THIS COMPETITION OR
HEME TUG OF WAR OCCURS IN THE
SAME INTRA CELLULAR COMPARTMENT.
WE HAVE DONE A HIGH THROUGH PUT
DRUG SCREEN TO BE ABLE TO
IDENTIFY COMPOUNDS IS.
>> CONCENTRATIONS TO TARGET LHR
WITHIN THE HOST MACROPHAGES
WITHOUT KILLING THE HOST CELL.
SO, ULTIMATELY, THERE ARE SOME
REALLY OUTSTANDING QUESTIONS
THAT WE WANT TO ANSWER.
THESE ARE NOT OUTSTANDING AS AN
AMAZING QUESTIONS BUT OUT STAND
BEING BECAUSE THEY STILL NEED TO
BE ANSWERED.
HOW IS HEME HOMEOSTASIS
REGULATED AT THE ORGANISMAL
LEVEL.
WE'VE BEEN ABLE TO GET SOME
INFORMATION AT THE CELLULAR
LEVEL AND SOME INFORMATION AT
HOW MULTIPLE CELLS COMMUNICATE
BUT HOW DOES THE ENTIRE ORGANISM
DEAL WITH THIS?
IS THERE A HEME SENSING
MECHANISM?
WHAT IS THE PATH OF
PHYSIOLOGICAL ROLE IN HUMANS AND
WE ARE STILL LOOKING FOR HUMAN
MUTATIONS THAT IF THEY ARE
THERE.
HOW IS HEME ACQUIRED AT THE OFF
THE HOST-PARASITE INTERFACE AND
HOW DOES HEME TRAFFIC IN LIVING
CELLS AND EARLY EMBRYOS.
WHICH WE REALLY DON'T UNDERSTAND
AND WE'RE ADDRESSING IT BY USING
GENETIC SCREENS IN C. ELEGANS TO
BE ABLE TO LOOK AT HOW ORGANS
AND TISSUES COMMUNICATE AND WHAT
IS THE PATH OF PHYSIOLOGICAL
ROLE IS THERE ALL THE HUMAN
VARIANTS WHICH ARE GENETIC
MODIFIERS OF DISEASE AND SUCH AS
SICKLE CELL, PLASMODIUM
INFECTIONS IT WAS DESCRIBED A #
HUNDRED YEARS AGO CALLED AFRICAN
EYE ON OVERLOAD IS THIS THE
GENETIC OF BOFTO SEE ROW SIS AND
HOW IS THE INTERFACE AND CAN YOU
USE SMALL MOLECULE INHIBITORS
AND FINALLY CAN WE DEVISE HEME
SENSORS AND NON-INVASIVE LABEL
FREE IMAGING TO BE ABLE TO LOOK
AT HEME AS ITS MOVING ON BETWEEN
CELLS AND WITHIN CELLS?
WITH THAT, THESE ARE THE AMAZING
GROUP OF PEOPLE THAT HAVE BEEN
DOING ALL THE WORK AND WITHIN
THING THAT YOU WILL NOTICE FROM
PHENOMENAL SET OF COLLABORATORS,
THAT NUMBER OF PEOPLE THAT HAVE
COLLABORATED AT THE NIH.
THEY HAVE BEEN VERY GENEROUS
WITH THEIR TIME, WITH THEIR
RESOURCES, AND HAVE DEEPLY
APPRECIATED THEIR HELP OVER THE
LAST 20 YEARS AND WITH THAT, I
WILL BE VERY HAPPY TO ANSWER
QUESTIONS.
THANK YOU FOR YOUR TIME AND FOR
YOUR ATTENTION.
[APPLAUSE]
>> GREAT AS USUAL.
I ALWAYS LEARN A LOT.
IF I REMEMBER THE SLIDE WITH THE
LEISHMANIA, IF YOU PUT THAT
LEISHMANIA INTO YOUR HRG1,
MINUS, MINUS MICE WHERE THE
HEME, TUESDAY THAT MAKE IT
RESISTANT TO LEISHMANIA?
>> OK, SO, THEY'VE BORDANE ASKED
ME THIS QUESTION IF YOU ARE
INFECT HRG1 KNOCK OUT MICE WHICH
IS ACCUMULATING A TON OF HEME AS
HEMA SEW INWITH LEISHMANIA, WHAT
WOULD HAPPEN?
SO, MY REGISTRANT MELISSA PERRY
HAS BEEN DOING THESE EXPERIMENTS
AND THE ANSWER IS, THAT THEY
HAVE VERY LOW PARASITE LOWER.
IF YOU TAKE THE SINGLE KNOCK OUT
PARASITE WHICH IS QUITE
CRIPPLED, AND INFECT THEM.
THEY DO WORSE THAN WILD TYPE
PARASITE.
BUT IF YOU LOOK AT THEIR ORGANS
THAT ARE INFECTED, IT'S A SIGN
OF THE MOR PARASITES YOU SEE IN
THE KNOCK OUT WHEN YOU INFECT
THEM WITH LEISHMANIA BUT WE'RE
TRYING TO FIGURE OUT WHY THAT IS
THE CASE.
>> ARE THE MA MALIA CELLS MAKING
IT AND FOR THUR USE AND A CREM
CELLS HAVE A REFERENCE OR DO YOU
THINK IT'S EVOLVED IS A
PRODUCTIVE MECHANISM.
>> I'LL COME TO A POINT WHERE I
DON'T KNOW THE INVESTIGATOR.
[LAUGHTER]
>> BUT ASHLEY ASKED ME THIS
QUESTION THAT DO MAMMALIAN CELLS
MAKE A CHOICE THAT THEY CAN MAKE
THEIR OWN HEME AND THEY WANT TO
AND WHERE THEY CAN TAKE UP THE
HEME FROM THE OUTSIDE WHEN THEY
HAVE TO?
SO I'M GOING TO ANSWER THIS
QUESTION IN COLLABORATION WITH
MY COLLEAGUE AT MOUNT SINAI AND
WE KNOCKED OUT THE HEME
SYNTHESIS PATHWAY AND PARADISE
AND THIS IS UNPUBLISHED, RIGHT.
THE INITIAL THINKING WAS HEME
SYNTHESIS IS SO CRUCIAL THAT YOU
ARE KNOCK THEM OUT THE CELLS
SHOULD NOT GO THROUGH ONE ROUND
OF REPLICATION BUT WHEN DO YOU
IT IN VIVO USING LOCKS P AND
CREE EXCISION, ASK YOU KNOCK OUT
HEME SYNTHESIS THEY HAVE NORMAL
HEME IN THE PARASITES.
AND I'M GOING TO MACK A LONG
STORY SHORT OF DOING EXPERIMENTS
OVER THE LAST TWO AND A HALF
YEARS AND THE SHORT ANSWER IS
THAT, THEY GET A SIGNIFICANT
PORTION OF THEIR HEME FROM KUPER
CELLS.
SO, I HAVE EVOLVED WITH THIS
THINKING NOW IN THE LAST SIX
MONTHS, THAT MAMMALIAN CELLS ARE
QUITE PLASTIC AND THE IF THEY
NEED TO MAKE HEME, THEY WILL
MAKE IT AND THEY NEED BEYOND A
SPECIFIC THRESHOLD, THEY'LL GET
IT FROM THE SURROUNDING CELL AND
WHAT BETTER CELLS TO GET IT FROM
THAN A PROFESSIONAL HEME
RECYCLING CELL SUCH AS A
MACROPHAGES.
SO IN ANY SENSE I THINK THAT
THIS IS EVOLVING INTO SOMETHING
WHICH C. ELEGANS HAS DECIDE A
LONG TIME AND THE EME CODE IS
44431.
OK.
>> IT'S A MOLECULE THAT WOULD
NEED TO BE SHAPER OWNED.
I'M CURIOUS, DO YOU THINK THAT
WE HAVE A SENSE OF THE GAMUT
THAT THEY UTILIZE OR DO YOU
THINK THERE WILL BE PRIZES THERE
AND I'M CURIOUS TO KNOW YOUR
GENERAL THOUGHTS ABOUT THIS.
>> DO I HAVE A SENSE OF THE
GAMUT?
I DON'T HAVE A SENSE.
PARTLY BECAUSE I WOULDN'T HAVE A
SENSE 15 YEARS AGO AS A HEME
TRANSPORTER BUT DOES IT MAKE
BIOCHEMICAL SENSE THAT THEY
SHOULD HAVE ONE, ABSOLUTELY.
AND I GIVE THIS EXAMPLE A LOT,
ACTUALLY.
THAT WHEN CHOLESTEROL SYNTHESIS
PATHWAY AS YOU SEE
SYSTEMATICALLY DISSECTED PEOPLE
SAID THERE'S NO REASON TO
TRANSPORT CHOLESTEROL, SINCE
THEN, INVESTIGATORS ARE
IDENTIFYING AN INTRICATE NETWORK
OF CHOLESTEROL TRANSPORT
PATHWAYS AS I WOULD CONSIDER
COLLEST TO BE INNOCUOUS COMPARED
TO HEME AND CHOLESTEROL, IF YOU
ARE KNOCK OUT THE PRE TEEN WHICH
WILL BE ALMOST EQUAL TO HRG1,
WALL IT NCP1, THE CHOLESTEROL
WITHIN THE MACROPHAGES AND
WITHIN THE LOOM INCONTAINS MMPC2
WHICH IS A CHOLESTEROL SHAPER
OWN WHICH TAKES UP AND DELIVERS
IT TO MCP1 SO IF THE CHOLESTEROL
CAN HAVE A CHAPERON AND A
TRANSPORTER, YOU WOULD BET THAT
THERE WOULD BE A HEME CHAPERON
WITHIN THE LOOMIN OF THE LIME SO
SEEM TO PICK UP THE HEME,
DELIVER IT AND THAT HEME THEN
SHOULD BE ROW LAID FROM HRG1 TO
EVEN OF THE DOWNSTREAM FACTORS
SO WORE DOING THAT IN VIVO USING
THIS MICE THAT WE DEVELOPED IN
THE LAST TWO YEARS.
>> THE QUESTION IS ABOUT THE YOU
KNOCKED OUT THE HEME PATHWAY IN
THE HIGH PATHO SIDES SO IS HRG1
UP REGUL REGULATED THERE OR IS 1
GOING TO THE M PLASMA MEMBRANE.
>> SO, WIN ASKED ME THIS
QUESTION THAT IF YOU LOCK AT THE
PARASITES THAT IS NOW, THAT CAN
CANNOT MAKE HEME, IS HRG1 UP
REGULAR GATED AND I DON'T KNOW
THE ANSWER BECAUSE WE JUST GOT
THESE RESULTS FROM THOSE.
BUT ALL I CAN CAN SAY THAT HRG1
FROM MACROPHAGES IS IMPORTANT
FOR SITES TO GET IT HEME AND I
DO NOT WHETHER IF IT'S IMPORTANT
OR NOT AND THE ONLY WAY TO DO
THAT EXPERIMENT NOW AND IS TO
KNOCK OUT HRB1 AND TO THE BONE
MR. OWE --
>> CAN YOU COMMENT ON
TRANSPORTING?
>> BY HRG1?
>> YES.
>> I CAN HAND WAVE AND I CAN SAY
THIS AND THAT THERE'S A HISS TA
TEEN IN THE CENTRE CELLULAR LOOP
WHICH IS CRITICAL FOR TRANSPORT
SO IT GOES FROM EXTRA CELLULAR
INTO THE CYTOPLASM IS IN THE
TRANS MEMBRANE DOMAIN IF YOU
KNOCK IT OUT AND THE TRANSPORT
DOESN'T HAPPEN AND THERE ARE
THROW RESIDUES IN THE TAIL WHICH
IS ESSENTIAL FOR HEME TO GET
OUT, LET'S CALL IT A CHANNEL FOR
THE SAKE OF ANYTHING ELSE.
TO GET OUT OF THE CHANNEL INTO
THE (INAUDIBLE).
IT'S COUPLED TO A V TYPE ATPAs
SO IF YOU KNOCK OUT THE V-TYPE
THEN H RG1 CANNOT TRANSPORT
HEME.
IT'S A SECONDARY TRANSPORTER.
I DO NOT KNOW ANYTHING ABOUT
WHAT ITS STRUCTURE IS.
IT RUNS MINIMALLY AS A TRIMER
THAT COULD BE A TETRAMER.
>> ARE YOU TARGETING THAT
DOMAIN?
>> I AM.
>> YOUR INHIBITOR.
>> CAN YOU COMMENT ON YOUR --
WHAT DOES IT LOOK LIKE?
>> CHEM BEING TREE, I'VE BEEN
WORKING WITH THE CHEMIST AND ALL
I CAN CAN TELL YOU AND THIS ALL
THE BLOCK AND OUR INITIAL GOAL
AND I'M GOING TO FINISH BY
SAYING IF ORGANISMS CAN TOLERATE
10 OR 20 FOLD BY SEQUESTERING
AND IT'S POSSIBLE TO USE AN
TAGGISM OF HRBBT AS A WAY.
>> WE HAVE A BASIC QUESTION.
WHAT IS THE BASIS OF HEME
TOXICITY AND WHY HAS THE BODY
CHOSEN TO TRANSPORT IRON IN A
TOXIC MOLECULE?
>> WHY HAS THE BODY CHOSEN TO
TRANSPORT IRON IN A TOXIC
MOLECULE?
OH, I SEE.
I AM WITHIN HEME AS A TOX HE CAC
MOLECULE.
IRON CLUSTERS COPPER, HEME SO IF
YOU WERE TO LOOK FROM AN
EVOLUTIONARY PERSPECTIVE, HEMES
HAVE EVOLVED MUCH LATER IN LIFE
THROUGH EVOLUTION THAT ARE
INVOLVED IN OXYGEN TRANSPORT.
CRABS AND LOBSTER DON'T HAVE
HEMOGLOBIN BASED.
I LOOK AT IT.
>> I'M TRYING TO PLAY THE PART
AS WELL.
>> SO I THINK THAT THE
REACTIVITY AS THE OXYGEN
CONCENTRATION INCREASED IN THE
ATMOSPHERE, HEMES ARE FAR MORE
CATALYTICALLY SUPERIOR THAN
OTHER FORMS OF FACTORS.
>> SO WHY HAS THE CELL CHOSEN TO
TRANSPORT HEME INSTEAD OF NON
HEME BASED CO FACTORS.
I CAN CAN REALLY HAND WAVE.
I DON'T HAVE FULL ANSWERS FOR
THAT, ALLEN.
>> THANK YOU THAT YOU'VE
INSPIRED AT LEAST A DOZEN PEOPLE
TO JOIN WALS LISTSERV