Pathology Grand Rounds: October 20, 2022

October 20, 2022

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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&amp;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&amp;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&amp;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.