Pathology Grand Rounds: October 20, 2022
October 20, 2022Information
The Synthesis and Dynamic Landscape of tRNA Epitranscritome presented by Patricia Dos Santos, PhD
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- 00:00OK. Good afternoon and welcome to
- 00:04today's grand rounds in pathology.
- 00:09I would like to introduce today's speaker,
- 00:13doctor Patricia Dossantos.
- 00:15A professor and associate chair of
- 00:18chemistry from Wake Forest University.
- 00:22Patricia received her bachelor's
- 00:24at the Federal University of Rio
- 00:28Grande in Porto Alegre, Brazil.
- 00:30And then came to the United States to do
- 00:36a PhD in biochemistry at Virginia Tech.
- 00:40After completing her PhD,
- 00:42she stayed at Virginia Tech for
- 00:45three years as a postdoc associate.
- 00:47And was then recruited to the
- 00:50Department of Chemistry at Wake Forest
- 00:52University as assistant professor,
- 00:54where she very quickly wrote through
- 00:57rose through the ranks to full
- 00:59professor and associate chair.
- 01:01Throughout her academic development,
- 01:04Patricia has always been outstanding.
- 01:07In the year she graduated with her doctorate,
- 01:11she received the Outstanding Graduate
- 01:13Student Award at Virginia Tech,
- 01:15and she was also the commencement speaker
- 01:19at the graduation ceremony at Virginia Tech.
- 01:23She further received at Wake Forest the
- 01:26faculty Excellence in Research award,
- 01:29the Robert Depth and Deborah
- 01:31Lee Faculty Fellowship Award,
- 01:33and the Eureka Faculty Award of
- 01:36Excellence in Mentoring and Research.
- 01:38Patricia has published over 60 peer
- 01:41reviewed papers and book chapters,
- 01:44she's an editor in methods in molecular
- 01:47biology and she has had close to
- 01:50150 invited talks and peer reviewed
- 01:52posters and platform presentations at
- 01:55national and international meetings.
- 01:58She has been funded by two grants from
- 02:01by the National Science Foundation and
- 02:04has interdisciplinary research grants
- 02:07from the North Carolina Biotechnology Center.
- 02:12In addition to her excellent science,
- 02:14Patricia is a extremely successful
- 02:17teacher and mentor.
- 02:19She teaches 6 biochemistry courses
- 02:21at Wake Forest.
- 02:23She has mentored 20 PhD students
- 02:28and honor students.
- 02:31She has also mentored 10 postdoc Fellows,
- 02:34and she has a fantastic track record
- 02:39of mentoring undergraduate students.
- 02:41Who do their honour research
- 02:44thesis in her lab.
- 02:46So she has mentored over 60 of
- 02:50these undergraduate students.
- 02:51Outside of Wake Forest,
- 02:53she is a reviewer on standing
- 02:56review panels for NIH,
- 02:58National Science Foundation
- 02:59and the Department of Energy.
- 03:01She has been a session chair at
- 03:04Gordon Conferences and a session
- 03:06chair at International Conference
- 03:08for iron sulfide clusters.
- 03:10She's an epoch reviewer for many national
- 03:14and international funding agencies.
- 03:16Her talk today is entitled The
- 03:19Synthesis and Dynamic Landscape of
- 03:22Transfer RNA's Epic transcriptome
- 03:24and I think we are in for a treat,
- 03:26so please welcome Patricia Dossantos.
- 03:33Thanks for the kind introduction
- 03:36and invitation to come here.
- 03:39I had a really great time.
- 03:41Today very special meeting and
- 03:43talking about women in academia.
- 03:45Really enjoyed that with
- 03:48department colleagues.
- 03:51OK. So what I want to share with you today,
- 03:55it's my work on T RNA modification.
- 03:58So in changes on T RNA modifications
- 04:02and how that has an impact,
- 04:05but before I do that kind of
- 04:07like very broad, we know that.
- 04:11Several biochemical reactions,
- 04:12they rely on the chemistry afforded by
- 04:15protein cofactors and then the enzymes
- 04:18associated with those cofactors.
- 04:20So among those cofactors,
- 04:22sulfur containing cofactors are widely
- 04:24distributed in nature and then they are
- 04:27actually participate in several lives.
- 04:28Sustaining reactions like photosynthesis,
- 04:32respiration, Nigel fixation will not
- 04:35be here today without those processes.
- 04:38So the main purpose of my lab is really.
- 04:42Trying to understand.
- 04:44Chemical futures for the synthesis
- 04:47of sulfur containing cofactors,
- 04:49which are some of the molecules
- 04:50shown here on this screen,
- 04:52and so scientists and the
- 04:54function of those four factors,
- 04:56so relevant for today's talk,
- 04:58is the synthesis of Taiwan nucleosides.
- 05:04You see them out, yeah.
- 05:05So Tyrone nucleosides that
- 05:06are present on to your RNA.
- 05:11So T RNA has, you know,
- 05:14very well known function as serving
- 05:16as a dactyl in translation.
- 05:19Granada reacts with aminoacyl tyranny
- 05:23synthetase for the attachment of amino
- 05:26acids to their cognate T RNA molecules
- 05:29which are then brought to the ribosome
- 05:33at the ribosomes tier and a interact
- 05:36with the messenger RNA enabling the
- 05:38translation of the genetic code and
- 05:41the by doing so they promote peptide
- 05:45synthesis or protein synthesis.
- 05:47What most people don't know is that tyranny.
- 05:50One, no canonical role,
- 05:52so like several roles in Atable ISM,
- 05:55so tyranny molecules,
- 05:56they have an impact on gene regulation.
- 05:59They are also able to sense nutrient sensing.
- 06:02So the relevant to the story that
- 06:04I'm going to share with you today,
- 06:06they are mediate a seller
- 06:09and stress responses.
- 06:10They can sense UV radiation and
- 06:13modulate translational capacity.
- 06:15Under those conditions T RNA is
- 06:18important for viral replication.
- 06:20So the example here is that
- 06:22modified T RNA actually assists
- 06:25replication of the HIV virus.
- 06:27And they're involved in other
- 06:29functions in addition to the
- 06:32roles of intact tyranny molecules.
- 06:34In all these processes that I described
- 06:36to you, fragments of tyranny a,
- 06:39there are also equally important
- 06:40in metabolism. They are involved.
- 06:43They sell proliferation in cancer,
- 06:45it hosts defense mechanisms.
- 06:47So fragments of that tyranny is not only
- 06:52byproducts of degradation of T RNA.
- 06:55Uh.
- 06:56So.
- 07:28The consequences?
- 07:32OK. I'll keep going for those of you there.
- 07:39Read tyranny modifications
- 07:42reported into biological TRN's.
- 07:45So in the human genome there are
- 07:47many T RNA genes and then we
- 07:50what we know is that ternative is
- 07:52found in the cytoplasm and called
- 07:54by nuclear genomic information.
- 07:57It's also modified.
- 07:59So while the structure of tyranny
- 08:01and then another subset of
- 08:03modifications are found on T RNA
- 08:05that is mitochondria encoded to RNA
- 08:07and those modifications are are.
- 08:09Important for translation at
- 08:11the above processes.
- 08:14So I typically don't talk a
- 08:17whole lot about pathology,
- 08:18but I thought this crowd would be
- 08:21interested to know that a Baron or
- 08:24altered accumulation of tyranny
- 08:26modifications are are associated with
- 08:29a variety of pathological disease.
- 08:32So we have mitochondrial disease
- 08:34that are associated with mutations
- 08:36on enzymes that are involved in the
- 08:38synthesis of a T RNA modifications
- 08:40in the mitochondria you have
- 08:42disease that are affect.
- 08:44Neurological defects associated
- 08:47with cancer and even diabetes.
- 08:50So the slide is really small.
- 08:53So the intention here is not for you
- 08:55to read all the disease that are found,
- 08:57but you can read more about
- 08:59this in this Suzuki paper.
- 09:01This is not my my literature,
- 09:03but it's a paper from a leading
- 09:05scientist on the ****** tyranny
- 09:07modification field and you can get a
- 09:09good idea of the variety of metabolic
- 09:13consequences associated with this.
- 09:15Function of T RNA modifications.
- 09:18So in a recent review did on in
- 09:21Dagley has stated that dysfunctional
- 09:23protein synthesis at the level
- 09:25of translation elongation,
- 09:27so at point where T RNA modifications
- 09:30become relevant is now recognized as
- 09:33a major pathophysiological driver in
- 09:36many human disease including cancer.
- 09:39So this topic of research is really
- 09:42important and and oftentimes
- 09:44overlooked one understanding.
- 09:46A disease phenotype and molecular level.
- 09:51So the modification of interest that
- 09:53I want to share with you about is a
- 09:55modification that it's a 2 by your audience.
- 09:58It's a modification that involves
- 10:00sulfur and that you know that's
- 10:02kind of it's fits into the whole
- 10:04umbrella of my research program
- 10:05that we will understand how sulfur
- 10:07containing cofactors are assembled.
- 10:09So this modification is found at
- 10:12the wobble position of tyranny,
- 10:15so interacts with the last space
- 10:17on the codon and it's phylogenetic
- 10:19conserved in all three.
- 10:21Domains of life and it's present
- 10:23in terminating codes for glutamine,
- 10:25lysine and glutamate.
- 10:27And the importance of this modification
- 10:30is that introduction of a soul for a
- 10:33two position of uridine ring allows
- 10:36canonical base pair formation with
- 10:38adenine for let's say AAA cordon of lysine.
- 10:42But also taught totalization of
- 10:45this modification allows for non
- 10:47canonical base pair formations.
- 10:50So in this case the you can base.
- 10:52Pair with the G so lack of those
- 10:56modifications that causes, you know,
- 10:59severe metabolic consequences.
- 11:01What it's what it's known that is inhuman.
- 11:03This modification is found both in the
- 11:06cytosolic T RNA is in the mitochondrial DNA.
- 11:10Interestingly,
- 11:11the biosynthetic pathway to modify
- 11:14terminating the cytosol is different
- 11:16than the one that is used to synthesize
- 11:202 thymidine in the mitochondria.
- 11:22And the mitochondria pathway is similar
- 11:24to that of what it's found in bacteria
- 11:27so reinforces the idea that you know,
- 11:30bacteria was a ancient microorganisms
- 11:32that had been gold to into ****** cells.
- 11:36So here's some good examples of
- 11:39biosynthetic enzymes involved in the
- 11:41cities of two pyridine and mutations
- 11:44have been found in those genes
- 11:46and they are diseased phenotype.
- 11:48So I'm not really.
- 11:52A sanitise that studies human disease,
- 11:55but that's my effort to to connect with
- 11:57the audience by primary interest is really
- 12:00trying to understand bacterial metabolism.
- 12:02And what we know is that in bacteria
- 12:05lack of two pyridine or mutations in the
- 12:09the biosynthetic components of tooth
- 12:11iodine leads to compromise cellular
- 12:13viability and in some cases including the
- 12:17organisms that I am interested about,
- 12:19this pathway is fully essential
- 12:21for the organization. Survive.
- 12:23OK, so it's it's an essential
- 12:25cellular process,
- 12:26which makes very interesting if you think
- 12:29about pathogenic bacteria and how you
- 12:32can discover new metabolic interventions for.
- 12:37For the treatment of infections
- 12:40caused by pathogenic so in the
- 12:43synthesis of new antibiotics.
- 12:45So that's the story that I
- 12:48wanted to tell you about how?
- 12:51My students and I went on this mission
- 12:54to really understand and identify
- 12:57the biosynthetic components of two
- 12:59pyridine and Bacillus subtilis,
- 13:02and in the work that we have done more
- 13:04recently to uncover the additional
- 13:06functions that this modification
- 13:08may be playing a role in,
- 13:10in this particular Organism.
- 13:12So that's the work of two very
- 13:15talented graduate students in my lab,
- 13:17I, Catherine Black,
- 13:18and actually Edwards actually has graduated.
- 13:21Uh, last month.
- 13:22So she she's now off to do bigger
- 13:25and better things in her life,
- 13:26but I'm grateful for the discoveries
- 13:28that she made here.
- 13:29So there are three main points
- 13:31that I want to convey here on our
- 13:34study of two pyridine biosynthesis.
- 13:36It's our experimental approach
- 13:38to identify biosynthetic enzymes,
- 13:41the function of two,
- 13:42two pyridine as a marker or potentially
- 13:45sensor of so far availability in
- 13:49Bacillus subtilis and how we.
- 13:51Understand the biochemical principles
- 13:53by which those enzymes operate that
- 13:57restrict and direct their roles
- 14:00in the sulfur metabolism.
- 14:05Before I dive in as that,
- 14:06I wanted to kind of like show you some
- 14:09key points so you can have an appreciation
- 14:12for the complexity of studying those
- 14:14systems at the biochemical level.
- 14:17So what we know is that in the centers of
- 14:20Tyler cofactors and I mentioned before,
- 14:23I'm interested in the cities of all the
- 14:26biomolecules showing here on this slide.
- 14:28What we know is that the first staff
- 14:30on super mobilization is catalyzed by
- 14:32an enzyme called cysteine disulphide.
- 14:35So those enzymes they they use a ping
- 14:38pong mechanism to bind sustain and
- 14:41convert sustain into alanine and by
- 14:44doing so they form a covalent sulfur
- 14:48personified enzyme intermediate.
- 14:49The sulfur then is transferred to
- 14:52downstream pathway components involved in
- 14:55the synthesis of iron sulfur clusters,
- 14:57tail nucleosides or different vitamins.
- 15:01As in the case of tyramine, lipoic acid,
- 15:05biotin, so on and so forth.
- 15:08But it's a complicated here is that
- 15:11if you have at least in humans,
- 15:13you have one single enzyme,
- 15:15NFS one that is responsible for the
- 15:17synthesis of all time nucleosides
- 15:19in the human genome.
- 15:21So that's that enzyme is found
- 15:22in the mitochondria.
- 15:24In some study model systems like E coli,
- 15:27you also have a primary enzyme and then
- 15:31the sulfur transfer pathway here is
- 15:34shared across different pathways, right?
- 15:37So you have one.
- 15:38Primary sulfur donor then and then
- 15:40that sulfur is traffic to different
- 15:43pathway intermediates and in some
- 15:45cases those biosynthetic intermediates
- 15:47are shared as in the case of two five
- 15:49year adine and moco Biogenesis which
- 15:53is also a protein called factor.
- 15:57To complicate things a little bit more,
- 15:59is that one interest that I had for
- 16:03many years and I guess that's where
- 16:05most of my publications come from,
- 16:07is really trying to understand the
- 16:09pathway that leads to the synthesis
- 16:11of iron sulfur clusters,
- 16:12very essential protein cofactors.
- 16:14And then what we want to do is like
- 16:17when we try to deconvolute those
- 16:19pathways and try to understand
- 16:22metabolic defects associated with
- 16:24the initial sulfur mobilization step.
- 16:26It's quite complex because what we see
- 16:30is that there are so iron sulfur enzyme,
- 16:33so enzymes that depend on iron
- 16:35sulfur cluster for the synthesis of
- 16:38other sulfur containing metabolites.
- 16:40So when you disrupt the initial steps on
- 16:45sulphur mobilization so for instance.
- 16:48For the synthesis of tyramine,
- 16:50you don't really know if you're
- 16:52disrupting the primary route for sulfur
- 16:55transfer or if you're inactivating a
- 16:57biosynthetic enzyme that requires an
- 16:59iron sulfur cluster for its activity.
- 17:01So you have this intertwined metabolic
- 17:04pathways that you have dependency
- 17:06of a super containing cofactor in
- 17:09the biosynthesis of another super
- 17:13containing cofactor. OK.
- 17:16So one primary metabolic.
- 17:23A component that we look at it's
- 17:26tyranny and how T RNA is modified.
- 17:28And this is really great because
- 17:31on T RNA modification of a pool you
- 17:35can identify pathways,
- 17:37enzymes that contain iron,
- 17:38sulfur clusters and then they would.
- 17:44Transfer sulfur to the
- 17:46cities of this cofactor.
- 17:49So this is an example here.
- 17:50The mutations that deplete this
- 17:53modification causes diabetes,
- 17:55for instance, so and the enzyme that
- 17:58does that is an enzyme that contains
- 18:01an iron sulfur cluster you have.
- 18:04Sometimes I miss the mouse.
- 18:07You have pathways like the S2
- 18:09you pathway that do not require
- 18:12iron super enzymes and then you
- 18:14have empathy is not shown here
- 18:16that do not contain sulfur but it
- 18:19depends on iron sulfur cluster.
- 18:20So you can interrogate the cells
- 18:23under different conditions and
- 18:25you can extract the that tyranny.
- 18:27And the primary tool that we use
- 18:29here is to purify those two RNA
- 18:32molecules and then analyze the relative
- 18:34abundance of all those nucleosides using.
- 18:37High resolution mass spectrometry.
- 18:39So when we do that,
- 18:40we not only analyze the analyte of interest
- 18:43as as to you in this particular case,
- 18:46but we look at the relative levels of
- 18:48all those different modifications.
- 18:50This is important because for
- 18:52instance in the case of two tire
- 18:55reading that is in this box here this
- 18:58modification comes along with additional
- 19:00modifications on the uridine base.
- 19:03So if we really want to quantify the effects
- 19:05on the biosynthesis of two thymidine.
- 19:07We have to quantify all those
- 19:10different metabolites and understand
- 19:12the their relative accumulation.
- 19:16Umm. Another point to consider when
- 19:19pursuing this analysis is that the levels,
- 19:22the relative levels of tyranny,
- 19:23modifications, they vary with growth phase,
- 19:29growth conditions,
- 19:30temperatures and so on and so forth.
- 19:33So the notion that TNA is modified and
- 19:36now is fully functional to perform roles
- 19:39in translation is really misleading.
- 19:42What we know is that there are
- 19:44degrees of modification that
- 19:46confined to the functionality.
- 19:48Of that tyranny and therefore fine
- 19:50tune the efficiency of translation.
- 19:53So for instance,
- 19:55if you grow cells and the
- 19:58different temperatures,
- 19:59you can have a different loading
- 20:02of of modification and that's
- 20:04interpreted as improving the
- 20:06rigidity of that anticodon loop.
- 20:09So you can favor base pairing
- 20:12information even in a higher temperature
- 20:14as in the case of the MSU I6A.
- 20:19You can also see a differential
- 20:21accumulation of some modifications
- 20:23under oxidative stress and this is
- 20:25attributed because some of those
- 20:27modifications they are dependent
- 20:29on enzymes that contain iron
- 20:31superclusters and then those clusters
- 20:34are susceptible to oxidative damage.
- 20:36So you can have a different
- 20:38accumulation as a as a readout for
- 20:41oxidative stress in those cells.
- 20:46So, so when it's studying or trying
- 20:48to assign a different pathways in the
- 20:51organisms that have not been studied before
- 20:54the the standard procedure to do that,
- 20:57it's like you take a pathway that
- 21:00is well studied and established and
- 21:01then you start to blast for that
- 21:04gene in that particular Organism and
- 21:06then you find equivalent components
- 21:08that you can make a good assumption
- 21:10based on sequence analysis. So.
- 21:12So when we start that that research that's.
- 21:15Out of the office workflow to fish out those
- 21:19genes and and to do biochemical experiments.
- 21:22So but that's not really the case.
- 21:25So when we compare the pathway found
- 21:27in E coli that is where we study and
- 21:30then we're trying to find equivalent
- 21:33genes in the basal subtilis genome.
- 21:35We do not find a complete pathway
- 21:38in terms of all the enzymes are not
- 21:41really present in some enzymes are
- 21:44missing suggesting that.
- 21:45Now you have perhaps an alternate mechanism
- 21:49to synthesize that same cofactor.
- 21:52And that's the case here for two pyridine.
- 21:54So our attempts to search for all
- 21:58those enzymes here showing red fail.
- 22:01So that means that imbecile subtilis,
- 22:03you don't have those pathways like 5
- 22:06pathway components are completely missing.
- 22:08So how you go,
- 22:09how you go from the Sistine,
- 22:11the sulfur race to the final
- 22:13enzyme in the pathway was the
- 22:16question for that particular study.
- 22:18OK, So what we know is that in the collide.
- 22:22Those pathways are really well studied.
- 22:24There's one primary enzyme,
- 22:26there's three dominant sulfur receptors,
- 22:28and then sulfur receptors showing
- 22:30in blue are the ones that Channel
- 22:32the sulfur to the biosynthesis.
- 22:34So for example,
- 22:35if you delete a you get a very distinct
- 22:39phenotype that is a deficiency of
- 22:41two pyridine and deficiency of MOCO.
- 22:44But you don't affect other pathways
- 22:46in reality actually affect a little
- 22:48bit because you are disrupting the
- 22:50equilibrium or so for transfer.
- 22:51So you actually.
- 22:52That boost on iron sulfur production
- 22:54because you don't have that
- 22:56competition anymore?
- 22:59What we found, you know very early stages
- 23:01when I when I joined Wake Forest is that
- 23:05Bacillus subtilis doesn't contain one
- 23:07Sistine the sulfur is it contains 4.
- 23:09So that to us already told that you
- 23:14know some of those pathways were
- 23:16different and then by looking at the
- 23:18genome neighborhood we could get some
- 23:20insight about their physiological
- 23:22functions in their particular Organism.
- 23:25So so far in my lab and also others
- 23:28in the field.
- 23:29Have been able to demonstrate the
- 23:32partnership between assisting the
- 23:34Sofras and they're still perceptor
- 23:37and validate their proposed roles
- 23:39in the centers of tailcoat factor.
- 23:41So I have done work in some of
- 23:44those all those proteins,
- 23:45but what I'm going to concentrate the
- 23:48talk it's on wire VO&MA they're they're
- 23:52relevant for two though you're adding.
- 23:55So as a biochemist,
- 23:56the first thing that we do is
- 23:59actually isolate the existing the
- 24:01Selfridge perform enzyme kinetics.
- 24:02And some of those initial studies
- 24:05show very clearly that those enzymes
- 24:07display very distinct kinetic behaviors.
- 24:10What it was really interesting.
- 24:11So those initial assays were done
- 24:14like most people in the field do.
- 24:17So everybody at that point would
- 24:19do just have kinetic reactions
- 24:21where you react with sustain and
- 24:24then you measure the half.
- 24:26Reaction rate through
- 24:28quantification of sulfide.
- 24:33What we wanted to do is then
- 24:36demonstrate that the presence of
- 24:39the physiological sofa receptor,
- 24:41so the second service trading this
- 24:43reaction was a valid SOFA receptor.
- 24:47And then in this particular case,
- 24:48the reaction rate in the presence
- 24:51of the sulfur acceptor enhanced
- 24:53about over a hundredfold,
- 24:55so indicating that the sulfur here in
- 24:58this case was much better in abstracting.
- 25:02The personal file then
- 25:04an artificial reductant.
- 25:08Sure.
- 25:11Reaction.
- 25:15Yes, you.
- 25:20Yeah. So you may want to think there
- 25:23are very high but the intracellular
- 25:25level concentration on reduce this
- 25:27thing in the cells of his 68 micromolar.
- 25:31So actually that's actually quite nice if
- 25:35you study kind of like metabolism because
- 25:39most enzymes they kind of operate around
- 25:43the KM that they they have you know.
- 25:45So if the concentration is it OK
- 25:48that allows the cells to adjust.
- 25:51The velocity of that reaction because they
- 25:53are operating around KM and that's the case.
- 25:56But one thing that you may,
- 25:57you know, kind of notice is that the
- 26:00lowest KM here is for a wire VO.
- 26:03You know one thing that we may want
- 26:05to postulate like because this enzyme
- 26:07is so essential in this modification,
- 26:09so essential maybe the enzyme has
- 26:11evolved to have a really low KM
- 26:14to give a preference for,
- 26:16so for mobilization on that pathway
- 26:19under conditions that sulfur
- 26:20is not readily available.
- 26:25No, no, that's great.
- 26:30Guys.
- 26:32Top.
- 26:40Not this enzyme.
- 26:41The next enzyme on the pathways
- 26:44coupled to the hydrolysis of ATP?
- 26:47Yeah, not, not this particular.
- 26:50Not any assistant sufferers.
- 26:53They use POP chemistry to dissolve urate.
- 26:57And we have done, I have not included
- 27:00here extensive kinetic analysis to
- 27:02show this is a ping pong mechanism and
- 27:05the formation of the personal fide.
- 27:07It's a mandatory staff.
- 27:11So as you imagine,
- 27:12there's a kinetic burst in the
- 27:15absence of the Super receptor
- 27:17and then this enzyme actually can
- 27:20slowly decay to turn over here,
- 27:22but the presence of the software
- 27:24sapter enhances over 100 fold
- 27:26the overall catalytic cycle.
- 27:35Periodic cells. Similar.
- 27:39Yeah, it could excel.
- 27:41So sisting the cell phrase activity,
- 27:44it's confined to the mitochondria,
- 27:47so there's only one gene.
- 27:49And FS1, the activity of this enzyme
- 27:51is confined to the mitochondria.
- 27:54And interestingly,
- 27:55the reactivity of this enzyme is highly
- 27:58dependent on the presence of the sulfur
- 28:00acceptor and modulating proteins.
- 28:02So there's a sulfur mobilization
- 28:05just coupled to iron metabolism.
- 28:07So there's a protein.
- 28:09It's called for taxing the
- 28:11binds to the sustained,
- 28:13the suffrage and the binding of the
- 28:15frataxin to the assisting the surfaces
- 28:18enhances the rate of sulfur transfer.
- 28:21And then what it's new now is that.
- 28:25This reaction is dependent on
- 28:26the SOFA receptor where iron
- 28:28sulfur clusters you're bound.
- 28:29So relevant for pathological behaviors
- 28:32is there's a disease that's called
- 28:35Fredericks ataxia that's associated
- 28:37with mutations in the FRATAXIN
- 28:40gene and that disrupts not only
- 28:43iron sulfur metabolism but also
- 28:45disrupts iron metabolism overall.
- 28:48So mutation for taxing leads to
- 28:50mitochondrial iron overload at the
- 28:52same time you have all the phenotypes.
- 28:54Associated with iron deficiency
- 28:56because you are not channeling
- 28:58the iron to the proper places.
- 29:01Maybe just deviating, but you know,
- 29:04hopefully that answers your question.
- 29:06Yeah, OK.
- 29:09So what it's quite interesting is
- 29:11that those super receptor molecules
- 29:13are quite as specific,
- 29:15right.
- 29:15So you have this protein here
- 29:18stuff view then we have shown
- 29:20that in the zinc bound form,
- 29:22so tightly bound zinc,
- 29:23so the presence of so few enhances
- 29:26the activity of its partners so fast,
- 29:30but so few is not able to display
- 29:33similar behavior to other systems,
- 29:35the surfaces in Basilius as
- 29:37well as orthologs.
- 29:39Listing to sell for race for him
- 29:41to note here those enzymes are
- 29:43extremely similar so the E coli
- 29:45SUV S and Bacillus sub S they
- 29:48are over 60% identity identical,
- 29:50yet they cannot cross react so.
- 29:55And and that's something that it's
- 29:57an important feature of those
- 29:59enzymes to guarantee that the
- 30:01sulfur is channel to the proper
- 30:04pathway that you were recruiting.
- 30:06Umm.
- 30:08So that's not only a specific
- 30:11feature of self fast.
- 30:12So this wire the operating that
- 30:14we we eventually postulated the
- 30:17dead it's involving 2 pyridine
- 30:19has its activity enhanced by the
- 30:21presence of ATP and then we know
- 30:23that the catalytic competent form
- 30:25of 80 of MDMA that's higher delays
- 30:29is the ATP bound form.
- 30:31So the enzyme has to have ATP bound to
- 30:35be able to receive the sulfur and then.
- 30:39Follow the chemical reaction so we
- 30:42know that this happens out again
- 30:45through formation of a personal fight.
- 30:47Intermedia using those labeling
- 30:49so for 35 assays.
- 30:54What we also know is that you know
- 30:56what we are doing in vitro reactions is
- 30:58important to be mindful and critical
- 31:00about the reaction conditions and then
- 31:02whether those reaction conditions mimic
- 31:05physiological conditions in the cell.
- 31:07So like I mentioned,
- 31:09the field is populated with publications
- 31:12that use DT and that provides a
- 31:15means to quantify reaction products.
- 31:17So DT is none are available in the cell.
- 31:22So most cells use glucose.
- 31:23I also the activity of those
- 31:26enzymes in the presence of mutation.
- 31:29It's very distinct,
- 31:30yet you can see an enhancement here.
- 31:34Tell us uh so.
- 31:35But Tillis doesn't use glutathione,
- 31:37doesn't make glutathione instead
- 31:38uses basically PIOS or have a
- 31:41collaborator in my department that
- 31:43synthesize facility file for me
- 31:45facility always a very poor reduction
- 31:48is even inhibits the reaction in DT.
- 31:51But pyridoxine is a quite
- 31:54effective personified reductase
- 31:56and enhances greatly the rate of a
- 32:01personified formation and reduction.
- 32:03And this is staff, but not at this is staff.
- 32:06So that's what we think it's
- 32:08happening in the cell.
- 32:09This is not a unique feature of wire VOC
- 32:13reactions done with stuff you and so fast.
- 32:16Also show that in the kinetic
- 32:19profile here indicates that the rate
- 32:22of sulfur mobilization is coupled
- 32:24with the rate of sulfur reduction
- 32:27on those kinetic schemes.
- 32:29So next what we wanted to do it's
- 32:32kind of replicate what we're seeing
- 32:34in vivo or at least what we are
- 32:36postulating in in vivo that can we.
- 32:39So we know establish that where
- 32:41there was assisting the self rays
- 32:44and then imma is a sulfa receptor
- 32:46here in the ATP bound form.
- 32:48So next we wanted to proceed on the
- 32:51pathway and validate it and then in
- 32:53May it's in fact a tire regulates that
- 32:56can pass on the sulfur to the tier and a.
- 33:00So the reaction that is proposed here is
- 33:03that in the first step of the reaction,
- 33:05so in uridine you typically have oxygen
- 33:08here which is not a good leaving group.
- 33:11So the first step on the reaction is
- 33:13that ventilate the uridine acquisition 2.
- 33:16So the sulfur can directly attack leading
- 33:18to the formation of a tooth iodine.
- 33:21So when we have done those experiments,
- 33:24yes we can form 2 pyridine in the test tube.
- 33:28So validating that this is.
- 33:30As A2 pathway component,
- 33:31you don't need all the seven
- 33:34components that we're seeing in E
- 33:36coli in the pathway is reliant on
- 33:38the presence of a reducing agent.
- 33:40So we don't know what step of it reducing
- 33:44agent it's used but we postulated
- 33:46it could be a desktop or desktop.
- 33:49So you can have personal fight attacking
- 33:51the other related or you can have a
- 33:54local reduction of that personal fight
- 33:56so you have 3 sulfide attacking that.
- 33:58Nevertheless,
- 33:59so we know and we can validate that
- 34:03we can go from you to 2π or you.
- 34:07But I also mentioned to you that this
- 34:10modification occurs in conjunction
- 34:14with a modification at A5 position.
- 34:16So in this case M and M5 as as
- 34:20to you and in the literature was
- 34:23proposed that those two pathways
- 34:25were independent one to one another.
- 34:28So you could either titillate
- 34:30your routine 1st and then you
- 34:32can modify the five position or
- 34:34you can modify the five position.
- 34:37And then thiolate seconds.
- 34:39So what we did here is that we use a T RNA,
- 34:44a mixture of tyranny a that contain.
- 34:48Unmodified you and partially modify you
- 34:51and then we incubating the reaction
- 34:55and to our surprise we're not able
- 34:58to detect the synthesis of the fully
- 35:02modified M&M as to you only to thy urity.
- 35:06So that initial result suggested
- 35:09that maybe the pathways are non
- 35:13independent bifurcated pathways,
- 35:15maybe there was some sequentiality
- 35:17on that pathway.
- 35:18Somehow and then they was only
- 35:22recognizing the unmodified view.
- 35:24Um, we?
- 35:27Took advantage that the availability
- 35:29of a crystal structure of MMA
- 35:32from an ortholog Organism that
- 35:34had the tyranny intermediate,
- 35:37adenylate intermediate.
- 35:37So this is the position of distillation.
- 35:41And so we look at the active side
- 35:43and what it kind of kind of became
- 35:47a structured justification for
- 35:48our results is that are in close
- 35:51proximity here for this carbon 5
- 35:54there was a concern venue alanine.
- 35:57Uh,
- 35:57that kind of provide a rationale for
- 36:00why this partially modified tier and
- 36:02a was not reacting to form 2 pyridine.
- 36:05So we thought that the phenylalanine
- 36:08was provided an historical hindrance
- 36:10not restricting the partially modified
- 36:13tyranny from entering the active
- 36:15side and getting tired related.
- 36:17So kind of the obvious experiment
- 36:19is to mutate the enzyme and see
- 36:21if we open up that active side.
- 36:23Now we can feed a bigger substrate
- 36:25and that's exactly what we are
- 36:27able to demonstrate.
- 36:28Is that mutant, so 55155 in Bacillus,
- 36:34154 in E coli, but that's the residue.
- 36:37So if you open up now we can
- 36:41make M&M so the fully modified.
- 36:45Modification,
- 36:47right?
- 36:49Using this variant form so those
- 36:51results kind of postulate that at
- 36:54least in the source of challenge
- 36:56the pathway for modifications is
- 36:58sequential where you till late 1st
- 37:00and then you modify it A5 position.
- 37:04So you know this work on devices
- 37:07was very interesting and and
- 37:09established that but still subtilis
- 37:12uses a dedicated system to Socrates
- 37:14and a SOFA receptor to violate a
- 37:17tyranna at the U34 position and
- 37:20what we thought here was that the.
- 37:24This distinctive reactivity provided
- 37:26sort of an opportunity for alternate
- 37:29regulation of the pathways involved,
- 37:32so for mobilization and
- 37:33biosynthesis of thiol factors.
- 37:35So by meaning is that you
- 37:37have a different system,
- 37:39the surfaces here showing in yellow
- 37:42and perhaps regulating them at a
- 37:44different conditions can kind of
- 37:47regulate specific pathways and
- 37:49scenario that is completely different.
- 37:52Than organisms that only have one
- 37:53assisting the self race right?
- 37:55Like you have to have a different.
- 37:59Mechanism to regulate those different
- 38:00pathways.
- 38:01So the experiment that we set
- 38:02ourselves to do it,
- 38:03it's is if those are so sulfur
- 38:07containing
- 38:07pathways, do we see a differential
- 38:10regulation if we grow cells under
- 38:14low sulfur versus high sulfur?
- 38:16So what we did here was that we cultured
- 38:19both type associate solar cells under
- 38:22various sulfur concentrations and
- 38:24then we analyze the relative levels
- 38:27of those proteins using Western blot.
- 38:30And So what you can see here is the
- 38:33relative abundance of wire video is
- 38:36greatly enhanced under sulfur replete
- 38:38conditions versus sulfur depleted conditions.
- 38:43And the same is true for in the MA.
- 38:48So what we have observed that
- 38:51both components wire VMA,
- 38:53they had decrease abundance on on
- 38:55their sulfur limiting condition.
- 38:57So if that is the case and then the
- 39:00other enzymes are not having much
- 39:04expression what we thought is that
- 39:06under conditions of Christ sulfur
- 39:08concentration then this pathway can
- 39:10proceed and you get accumulation
- 39:12on the fully modified.
- 39:14On tyranny and then under
- 39:17sulfur depleted conditions,
- 39:18then you don't get as much
- 39:21as modified to your RNA.
- 39:23And that's exactly what we have observed.
- 39:25So from South culture under different
- 39:28conditions we can analyze the relative
- 39:32levels of those modifications using
- 39:34high rates in Ms and we can see a nice
- 39:37dose dependent effect on the relative
- 39:41accumulation of this modification.
- 39:43So put into kind of repeat what I said
- 39:47before is this is an essential pathway,
- 39:49an essential modification but
- 39:51you have a cellular contacts that
- 39:53you kind of vary in the degree.
- 39:55Of modification,
- 39:56and presumably you were making the
- 39:59tyranny less optimal for translation.
- 40:05So what we know also from the
- 40:07literature that under conditions
- 40:09that tyranny is hyper modified is not
- 40:12fully functional and an offer often
- 40:15hyper modified T RNA's target for
- 40:17degradation and northern blot analysis
- 40:20showed that tyranny that is carried
- 40:24this modification has reduced levels
- 40:27under sulfur depleted accommodations.
- 40:30By analyzing T RNA we can also
- 40:34interrogate the relative levels of
- 40:37modifications that depend on this thing.
- 40:39The software is not a wire video
- 40:42and modifications that presumably
- 40:43depend on on self assess and known
- 40:46to depend on ebz two other sustained.
- 40:49The software is in this Organism
- 40:51remain steady under those conditions.
- 40:55Yeah, because my lab has
- 40:57an expertise on iris.
- 40:58So for Biogenesis we also test the
- 41:01activity of three different iron
- 41:02sulfur enzymes to see if those
- 41:05conditions are affecting iron super
- 41:07metabolism and that remains the same.
- 41:09Whereas you know likewise enzymes
- 41:11that do not depend on iris
- 41:14superclusters also is an out there.
- 41:16So if it looks like that sulfur
- 41:19availability has a targeted effect on
- 41:22modulating the pathway involving wire VO.
- 41:26And MDMA and is not really
- 41:29disrupting other pathways.
- 41:31So, you know,
- 41:32in this particular study we
- 41:35interrogate sulfur availability and
- 41:37then this modification is known to
- 41:40be affected by other physiological
- 41:42conditions and we are now carrying on.
- 41:47Modification, you know,
- 41:48analysis of the relative levels on the
- 41:50different conditions and testing hypoxia,
- 41:53UV radiation,
- 41:54heat and cold and interrogating the
- 41:57whole effect transcriptome TNA epigen
- 41:59script home in Bacillus subtilis.
- 42:02We do believe that this is not a
- 42:04specific phenomenon to Bacillus subtilis,
- 42:06but it's also observed in other types
- 42:08of bacteria and we have preliminary
- 42:10data to show that and it's also known,
- 42:13well documented for different types of.
- 42:17Product sales and mammals that you
- 42:19also have some of the equivalent
- 42:21cellular responses.
- 42:24OK. So the so now it's like
- 42:26the last bit of story that I
- 42:29want to tell you about this,
- 42:30our study on why review MMA.
- 42:33So we know that those
- 42:36enzymes are very specific.
- 42:38The results from the biosynthesis
- 42:40and also sulfur metabolism project
- 42:43really informed us that other
- 42:45assisting the sulfur races in this
- 42:47Organism cannot pick up the job
- 42:49offer wire VO and rescue those
- 42:52phenotypes by meaning that wire.
- 42:54Video is a dedicated sustained
- 42:56self race to MMA that plays a
- 42:59role once you tie your routine and
- 43:02you know stuff asked for instance
- 43:05cannot be the sulfur source to MMA,
- 43:08neither all this other system
- 43:10the soul phrases.
- 43:11We also have in vivo and in
- 43:14vitro data to show that the.
- 43:17The dedicated role,
- 43:19it's actually mutual because so
- 43:22few cannot cross react with our
- 43:24view and so on and so forth.
- 43:26So we wanted to understand what are
- 43:29the structure features on those
- 43:31enzymes that are really regulating
- 43:34their physiological functions.
- 43:35So one way to approach that is
- 43:38actually to cost compare the
- 43:40closest ortholog to wire view which
- 43:43is the E coli ISS, so those two.
- 43:47Proteins there are about 63%
- 43:49similar to each other,
- 43:51but yeah they cannot cross
- 43:53complement in vivo.
- 43:55So by meaning that if we transform and
- 43:58express wire view and an E coli cell,
- 44:00I cannot rescue the phenotype
- 44:03associated with ISS.
- 44:05So this was kind of like part of
- 44:08a a initial work where we can
- 44:11do those cross complementation
- 44:13and expression of wires.
- 44:15The old the vessel assisting the
- 44:17sofras cannot react cross react
- 44:19with us A and rescue their pathway.
- 44:21But if we Co express both vessels
- 44:24operating in an ISS knockout of equal life,
- 44:27we can fully rescue.
- 44:29The same is true for admission in the MA.
- 44:33We can only rescue if we express.
- 44:36This pathway.
- 44:39At the same time,
- 44:40so that tells us there's a specific
- 44:43sulfur transfer from wire video to MMA,
- 44:46and that sulfur transfer event
- 44:49cannot happen as prevented somehow
- 44:52from wire reveal to Tuesday
- 44:54to rescue that ecoli pathway.
- 44:57So that kind of provide the premise for
- 45:00a series of biochemical experiments
- 45:02where we test the cross reactivity
- 45:05of those enzymes in the test tube.
- 45:08So again via video enhances the catalytic
- 45:11activity of its physiological partner,
- 45:15but it does not react with the ecoli
- 45:18ortholog and then the ecoli system.
- 45:21The suffrage can only have its
- 45:23activity enhanced in presence
- 45:25of its geological partner,
- 45:26the.
- 45:27Why are video doesn't display
- 45:31that phenomenon so?
- 45:32We know those enzymes are catalyzing
- 45:35the same chemical reaction
- 45:36which is sulfur transfer from
- 45:39cysteine to a sulfur receptor.
- 45:41And then we took advantage of a
- 45:44crystal structure that was available
- 45:46in of ecoli ICS in complex where
- 45:49Texas A and we match the residues
- 45:52that were at this binding interface.
- 45:56And we postulated that perhaps
- 45:59the lack of cross reactivity of
- 46:02the Bacillus assisting the sulfur
- 46:04ace in E coli was associated with
- 46:07a now third
- 46:08binding interface.
- 46:11So what we did here is
- 46:13to guide those analysis.
- 46:14We conducted a multi sequence
- 46:17alignment using several sequences of ice.
- 46:20So this is just an example here.
- 46:21But several sequences of organisms
- 46:24that contain ISS like enzymes and
- 46:26use staff as partners and in several
- 46:29organs that contain wire veal like
- 46:31enzymes and use and then MA as
- 46:34sort of an abbreviated pathway.
- 46:36And then we try to map residues that
- 46:38were shared within those groups but
- 46:40there were distinct between those two.
- 46:42Families of assisting the cell for assist.
- 46:44So although they have a high
- 46:46degree of similarity,
- 46:48there were some key differences in.
- 46:50I want to point here on this table.
- 46:52So what do you see is that?
- 46:56And it's very interesting is
- 46:58that some of the residues that
- 47:00are at the binding interface,
- 47:03we thus say for instance glutamate 48
- 47:07and 49 in ecoli had opposite charge
- 47:10in the wire video sequence and that
- 47:14provide the basis for a proposal
- 47:16that those proteins were not cross
- 47:19reacting because they were not having
- 47:22a complementary charge afforded by that.
- 47:25Surface.
- 47:26Also important to note here is that
- 47:30this binding interface between
- 47:33ISIS and Tus 8/6 as I remote.
- 47:38So the active site is here and you
- 47:40have a loop that swings into the
- 47:43active side and donates the sulfur.
- 47:45So the binding interface between
- 47:47the enzyme and the sulfur receptor
- 47:50occurs at remote site and actually
- 47:52at a different sub unit where
- 47:55the catalytic chemistry is.
- 47:56Company,
- 47:58OK.
- 47:59So the obviously experiment that
- 48:01we set up ourselves to do is like
- 48:04construct a library of mutants and
- 48:06trying to take wire video and convert
- 48:08into ISS like by modifying those
- 48:11select residues into residues that
- 48:14are present in ISS and see if we
- 48:17can expand the reactivity of this
- 48:20enzyme towards equalized so for acceptors.
- 48:24So we first passed the reactivity
- 48:26towards the native effectors and
- 48:28you know some of those residues
- 48:30here they lost the ability to
- 48:33engage with the native acceptor,
- 48:35but most importantly.
- 48:38By doing this music Genesis studies
- 48:41we could screen that some of
- 48:44those mutations so 44, so R44E.
- 48:49Why are VOC?
- 48:52The head contain equivalent mutation here.
- 48:54So when we mutated to look like ISS
- 48:59now we can engage in a reaction with
- 49:04pasta and have these activities
- 49:06stimulation to you know over tenfold
- 49:10on the catalytic turnover rate and
- 49:13this is another super receptor.
- 49:16I that we don't have a crystal
- 49:18structure for the complex,
- 49:20but when we mutate 4 residues at
- 49:22that particular binding interface,
- 49:24we observe a high degree of extenuation,
- 49:28so suggesting that we are kind of.
- 49:32Who were somewhat successful
- 49:34in engineering those enzymes to
- 49:36now adopt in a very intentional
- 49:39mariner expanded reactivity towards
- 49:42selected pathways.
- 49:43So to validate some of those findings,
- 49:47what we did here is that we.
- 49:50We wanted to determine whether or not
- 49:54those variant enzymes were able to
- 49:58partake role on ISS pathways in vivo.
- 50:02So to do that work we use a ISS knockout
- 50:06strain and we know that this
- 50:09is spraying is affected in its
- 50:12ability to make four thought errity
- 50:15in a pathway that depends on π,
- 50:18and it's also affected in a pathway.
- 50:21To make 2 thyroxine in a through
- 50:24through engagement with us a.
- 50:27When we express the wall
- 50:29type of stimulus enzyme,
- 50:31we don't really rescue those pathways,
- 50:33so this is relative levels to
- 50:36the wall type equalized strain.
- 50:38However, when we take the
- 50:40single point mutation R44E,
- 50:44what we're able to achieve is fully
- 50:47recovery of two pyridine pathway
- 50:50indicating that it took only one single
- 50:54amino acid substitution to convert via VOA.
- 50:57Into an ISS like enzyme in the pathway
- 51:02requiring to say those results are
- 51:03kind of in agreement with the system,
- 51:05the self race because that's the mutant
- 51:08that we could show activity stimulation.
- 51:10Likewise this wire deal quadruple
- 51:13mutant here could engage with I,
- 51:16I and recovery if not even more
- 51:19accumulation of 4th iritty.
- 51:21And that's a pathway that depends on thi.
- 51:24This mutant again was a mutant that
- 51:26showed a 20 fold activity stimulation.
- 51:29So we're really pleased with this and
- 51:33kind of struck by the results that
- 51:36you know it takes only one change
- 51:38for for a gain of functionality.
- 51:41We also look at in terms of
- 51:45growth phenotypes.
- 51:46So ISS is involved with so
- 51:49many cellular processes.
- 51:51Inactivation of ISS causes
- 51:54a severe growth phenotype.
- 51:56If we express the wall type,
- 51:58it doesn't really help,
- 52:00it actually makes worse.
- 52:01But expression of that single
- 52:04mutant partially recovers ISS.
- 52:07We didn't observe full recovery
- 52:09in relation to the wild type and
- 52:12that's actually expected because
- 52:15we're selectively recovering one of
- 52:17the pathways that ICS participate.
- 52:19So in the end, you know my background,
- 52:23other pathways involving ISS,
- 52:24they still depleted and contributing
- 52:28to growth,
- 52:29growth rates in this particular Organism.
- 52:34OK.
- 52:34So with that,
- 52:35I don't know how I'm doing with time,
- 52:38but the main take away points that
- 52:41I want you to kind of remember
- 52:44from from this talk is that T RNA
- 52:47performs multiple roles besides
- 52:49translation and those roles are really
- 52:51important that defects on enzymes,
- 52:54they modify T RNA and like some
- 52:56some folks in the field they
- 52:59call riders TNA writers,
- 53:00they are associated with multiple
- 53:03pathologies and cellular viability.
- 53:05That the abundance of those
- 53:07modifications are impacted by
- 53:09nutrient availability and then
- 53:11I showed you the example about
- 53:13sulfur concentration but also their
- 53:15environmental factors that fine-tuned
- 53:17the functionality of transfer RNA.
- 53:20Then in Bacillus subtilis at
- 53:22least those modifications they
- 53:25involve dedicated enzymes and the
- 53:28interaction between the assisting
- 53:30the surprises and they are sulfur
- 53:33receptor partners is a very specific.
- 53:35Event that directs sulfur to the
- 53:39pathways they're participating.
- 53:41And then with that,
- 53:42I want to thank the people that have
- 53:45done the work. I have a very young lab.
- 53:48You may recognize the last name here.
- 53:50So nick?
- 53:54It was a member of my lab, he graduated.
- 53:56But I I'm very thankful for all the
- 53:59students that, graduate and
- 54:01undergraduate students that and
- 54:04are in the lab doing hard work,
- 54:06and also my collaborators that.
- 54:11Very important for other projects that
- 54:13I have not talked to you about it.
- 54:15I'm also thankful for the National
- 54:17Science Foundation that has been mainly
- 54:20funded this this project and other
- 54:22projects in my lab since they started
- 54:25and then thank you for your attention.
- 54:31It's time for a public question.
- 54:36Chat.
- 54:42Yeah.
- 54:47Oh, I'm sorry. So so
- 54:51how do I close? You know the first point.
- 54:56The modification was carried
- 54:57in the non canonical function.
- 55:01Umm.
- 55:04OK. So let's do that. Uh.
- 55:09There we go. OK. Sorry.
- 55:14Curated.
- 55:17Partner talk. Are the modifications to the
- 55:21modifications of the tyranny that dictate the
- 55:23other the other problems like in viral?
- 55:28Yeah. Directs the.
- 55:32So you know that's a really great
- 55:35one because for example tyranny
- 55:37lysine and it's modified form is
- 55:39actually a primer for HIV replication.
- 55:42So that modification is really important.
- 55:45I think the work that I just showed
- 55:48you in its modified form and then the
- 55:50work that I kind of show you here
- 55:52also kind of sides to that because the
- 55:55unmodified form is targeted for degradation.
- 55:57So I think you know for you guys
- 56:00that you know the pathology and.
- 56:03You know, I think there's a great
- 56:04deal of appreciation on, you know,
- 56:06let's run next gene sequencing transcriptome
- 56:09and then getting a proteome analysis.
- 56:14I think would be great.
- 56:16You also have the eppi tyranny transcriptome
- 56:18in some of those disease phenotypes right?
- 56:21Like I bet you will be altered.
- 56:24There are different modifications
- 56:26like cuisine is one that it's highly
- 56:30evolved on nutritional status and
- 56:32in associated with a whole slew
- 56:35of different disease phenotypes.
- 56:37So. How they do so? One way is.
- 56:45Depending on the seller response.
- 56:48T RNA is playing a role
- 56:51because expression of genes,
- 56:53for example stress response.
- 56:55It is known that genes involved in
- 56:59stress response they have a codon bias,
- 57:02so codons that require TNA that is
- 57:05modified are necessary for translation
- 57:08of those of those proteins and evolving
- 57:12in stress response for diabetes,
- 57:15for instance the MS2I6A.
- 57:19What was known is that
- 57:21for insulin translation.
- 57:23You require you have a codon bias towards
- 57:26T RNA that carries that modification.
- 57:29So if you don't have the modification,
- 57:32then you're compromising translation of
- 57:35of the proteins that depend on that.
- 57:38Does that make sense?
- 57:40Yeah, good question.
- 57:48See any of the benefits
- 57:50that you described utilized
- 57:52development for material, yeah.
- 57:54So there there's a good understanding
- 57:57much more on the operatic front.
- 57:59But for a bacteria,
- 58:00I think there has been some attempts,
- 58:02especially for modifications that
- 58:05are essential to target the.
- 58:08To target those writers,
- 58:10like tyranny writers,
- 58:12as a mechanism to hold.
- 58:16So viability but that is
- 58:20still kind of in its infancy.
- 58:23There's a great deal of interest
- 58:25more recent one up maybe targeting
- 58:27the system itself races because they
- 58:30have they are so specific right.
- 58:33So if you can just drop the function of
- 58:35those sustained itself races and that's
- 58:37specific super transfer event then you
- 58:40selectively inhibit those enzymes.
- 58:42So I don't really do drug
- 58:44development you know my research.
- 58:46Like as you can tell is more
- 58:49kind of fundamental basic science
- 58:51understanding biochemical pathways.
- 58:52But hopefully you know they're being
- 58:56interested from pharmaceutical companies
- 58:58on talking about let's trying to
- 59:00find an inhibitor especially liking
- 59:02but still is and other gram positive
- 59:04because you have multiple enzymes and
- 59:06then they have very specific phenotypes.
- 59:08Can we find any inhibitor that binds to
- 59:11like only so fast that is found in Gram
- 59:14positive as a specialized drug development.
- 59:16So antibiotic that targets only
- 59:19grandparent grand positive pathogens.
- 59:21Yeah.
- 59:27Good, OK.