81 – Illuminating the dynamic architecture and regulatory mechanisms of SynGAP

Eric S. Underbakke, PhD

Dr. Underbakke’s bio

Dr. Underbakke is an Associate Professor in the Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology at Iowa State University. His research team investigates the dynamic structures of signaling protein teams that tune neuronal connections. To profile protein communications in action, the Underbakke lab employs integrative structural biology approaches, including mass spectrometry, chemical probes, and enzymology. Eric grew up in Iowa and earned a B.S. degree at Iowa State University. He pursued a Ph.D. in Biochemistry at the University of Wisconsin, Madison. He developed an interest in the structures of synaptic proteins as post-doctoral research fellow at the University of California, Berkeley and The Scripps Research Institute. He returned to Iowa State University to start an independent research group in 2015.

THIS IS A TRANSCRIPT ONLY:

hello and welcome to today’s webinar my name is Olga Bothe and I’m part of the team here at syngap research fund our presentation today is illuminating the dynamic architecture
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and Regulatory mechanisms of syngap and I have the pleasure to introduce today’s speaker Eric
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andrebaki Dr underbaki is an associate professor and the Roy J Carver Department of biochemistry
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biophysics and molecular biology at the Iowa State University his research team investigates
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the dynamic structures of signaling protein teams that two neuronal connections excuse
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me to provide to profile protein Communications in action the underbaki lab employs integrative
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structural biology approaches including Mass spectrometry chemical probes and enzymology
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Eric grew up in Iowa and earned BS at Iowa State University he pursued a PhD in Biochemistry at the
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University of Wisconsin in Madison he developed an interest in the structures of synaptic proteins
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as postdoctoral research fellow at the University of California Berkeley and left Scripps Institute
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he returned to Iowa State University to start an independent research group in 2015. A recorded
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version of this webinar will be available on the SRF website under webinars on the family
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menu and then by the end of this presentation you will have the opportunity to get the answers to
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your questions and we’d love to hear from you so please write your questions in the Q a below and for those of you just joining us welcome again our speaker is Dr and Eric underbaki and
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his presentation today is eliminating the dynamic architecture and Regulatory mechanisms of syngap
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welcome Dr andrebaki thank you so much for the introduction it’s a real pleasure to join you
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all here today I am going to pop up a slideshow so that I can guide you all through what my research
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team is exploring in terms of the Dynamics and structures of syngap and some of their
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signaling mechanisms so again thank you so much for this invitation I rarely get the opportunity
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to speak to a community of people so invested and active in pursuing new research to treat a
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really important issue so I guess um I will start off with a little bit of introduction
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um there we go I want to introduce my team the people who do this research alongside with me and
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we’re at Iowa State University in a biochemistry program we are largely protein biochemists and our
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work is focused largely on the um kind of detailed structures of the proteins and their interactions
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and how they work and this is a sampling of the people who started the project an undergrad in
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my lab named Sarah and a graduate student who has since graduated and is at the National Institutes
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of Health now Quinn Henson and these are some current people in my lab graduate students Tanya
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and Alexandria and a small team of undergrads who work with them as well and uh collectively they
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are exploring a number of different uh questions involved in mechanisms of synaptic signaling
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so uh as welcome mentioned in that lovely introduction um I have had a long-standing
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interest in how signaling systems work the processes by which cells sense their environment
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and respond and communicate between each other and this is a um major and complex topic uh
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because uh a lot of the proteins involved in these communication pathways are membrane Associated if
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not going through the membrane then they’re often clustered next to the membrane and these proteins
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tend to have a lot of regulatory features they’re often composed of multiple domains they often are
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very flexible and highly Dynamic and they are often at a Tipping Point of responsivity they’re
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not really just slammed on or off they’re kind of capable of tuning their activities over a wide
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dynamic range and so this makes them challenging targets for classical structure function studies
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for biochemists and there are many many important disease States associated with the dysfunction of
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these systems so they’re a real Frontier Topic in lots of scales of biochemistry cell signaling
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genetics and developmental biology so I’ve studied several different signaling systems in my career
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um most recently before starting my lab I as a postdoctoral researcher was studying
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the mechanisms of nitric oxide signaling and this is a really strange and unique signaling pathway
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that involves a enzyme making a gas molecule that can kind of diffuse between cells and communicate
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really locally and one of the locations in which this nitric oxide gas signaling is important is
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at the synapse and so this was my introduction to synaptic signaling and the signaling proteins
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of the postsynaptic density so when I started my independent lab I moved kind of laterally
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looking at the nitric oxide signaling proteins and their scaffolds and looking at the other
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signaling proteins that were brought into this kind of signaling cluster with these scaffolds
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so um I probably don’t need to reinforce this too much with this audience but the
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proteins involved in the synaptic signaling are primarily responsible for two kind of
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levels of signaling of cell communication one is the kind of primary release of neurotransmitters
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and then generation of an action potential but there’s a bunch of signaling proteins in the postsynaptic density especially that are involved in tuning that Primary Response
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adapting to patterns of signal over time and they are responsible for kind of making
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decisions if you will at a molecular level for whether or not this is an important and uh well
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trafficked neuronal Connection in which case it can be strengthened or whether it’s prone to noise
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and background in which case it can be kind of tuned down and so this is a really complex system
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involving a number of different proteins that are often connected right next to the membrane
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through interactions with scaffolds and one of the most prominent is of course syngap so
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um there is a wealth of studies and research going on looking at all sorts of different signaling
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proteins in these systems including kinases G protein signaling the neurotransmitter receptors
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themselves and of course the scaffolds and collectively these are really important targets
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of study because it’s just well understood in an era of personalized genomics that mutations that
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impact the functions of these are associated with a really wide range of neurological conditions
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some of which can be quite severe and some kind of kind of on a spectrum lead to neuro
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divergences and so there is all sorts of study happening all kinds of levels here to investigate
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how these can proteins communicate how they sense the trafficking patterns the signaling and action
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potential patterns of the synapse and we are building on a multiple decades-long quest to
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understand these systems and the researchers that are exploring these systems are coming
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at it from multiple I guess scales of complexity from my end of the world the protein biochemists
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and we are very focused on a kind of dissection approach where we take individual proteins one
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at a time and we explore the fine details of their structures and functions and that’s been
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uh very important and useful for establishing a baseline of what these proteins do in isolation
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at the very other end we have uh tremendous work that’s been done through the decades studying at
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a more organismal level or at the cell based level uh identifying the proteins and Pathways
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involved in long-term potentiation long-term depression and all the other factors going into neural communication and synaptic plasticity and so these are pretty well established
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approaches to studying the mechanisms neuronal communication and both are converging on a
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Frontier that’s not exactly new but is still in need of a lot of work and that’s kind of the the
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mesoscale organization of the proteins involved in this communication rather than focusing
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on individual proteins one by one there is a increasing attention to how these proteins form
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scaffolded networks higher order complexes and how these move and rearrange and respond to stimulus
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and so this has been a classical challenge of structural biology but happily we have a lot more
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tools available to us nowadays so that we can explore these bigger scale signaling complexes
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I want to take a step back though for a broad audience and kind of
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I guess review some perspective on why we care about the structures of proteins involved
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in neural communication this field in general is known as structural biology and it’s kind
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of a descriptive field we’re interested in understanding how the structures of really large macromolecules involved in biology how those structures underlie their functions and
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that’s kind of an adage in in my field structure leads to function and a real big goal of this is
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to take the kind of conceptual cartoons where we we describe how enzymes or RNA or any of the
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other macromolecules of Life how they kind of interact at a cartoon level and we want to go deeper and understand what those molecules look like at a molecular level and this has been
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really important and useful for understanding how they work and how we can address their functions
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and manipulate their functions either through pharmacological interventions or other means
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so uh I guess one important lesson that I like to share with students in my introductory classes
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is that the molecules of Life are just molecules they’re behaving the same with the same chemical
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principles as any other chemical outside that you might encounter in a high school chemistry lab
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one major difference though is that these macromolecules as the name would suggest macro they’re huge and they have architecture they have really big cavernous structures of
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pockets and nodes and projectiles and this shape complexity allows them to recognize each other
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recognize small molecules and transform small molecules with real Exquisite precision so these
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real big structures can interlock with Incredible specificity they can change each other’s shapes
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they can sense and respond to each other and they can modify each other and it’s through those
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recognitions and modifications that we can get signaling and communication at a molecular level
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it’s also of interest to people trying to modulate that communication because the giant shapes of
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these proteins or other macromolecules allows us to develop drugs that can kind of sneak in and fit
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in those pockets and tune their functions so this is a long-standing frontier goal of biochemists
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and biologists in general understanding the structures of these large molecules and I
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like to think about what that really entails because we kind of take it for granted that
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this is just something we can readily do nowadays because we’ve been doing it for 50 or 60 years
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but what we’re asking of researchers is to describe these really really huge molecules at
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a atomic level looking at the kind of physical location of all of the atoms and how they’re
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connected with bonds and I remember when I was in college in an organic chemistry class my professor
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walked us through the really inspiring work that Emma Fisher did to reveal the structure
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of glucose which is right here it’s uh an ornate and important molecule it’s the foundation of our
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metabolism and it took a long time to understand how its atoms were connected and how they kind
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of projected in three-dimensional space but nowadays to understand cell biology we’re
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actually looking at structures of proteins and this is just an average kind of small protein the glucose binding protein and you can see just how dizzyingly complex this is it’s just a Warren
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of Thicket of different bonds connecting the atoms of amino acids and they kind of come together with
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this Global architecture that’s able to do things like in this case bind to and recognize glucose
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but that’s not all there’s been a lot of focus on the structures and functions of the proteins that we can readily capture and use our tools to describe the three-dimensional structure but
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there’s a whole nother class of proteins that have intrinsically disordered regions and these
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are proteins that don’t actually adopt a static stable structure and I should say In fairness all
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proteins have Dynamics to a degree they kind of breathe they Flex they move but there are huge numbers of proteins that either are entirely disordered or have significant regions of disorder
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where the structures of these are whipping around and capable of moving a lot and so a colleague of
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mine Julian Roche calls this the kind of dark matter of the population of proteins in cells
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so we have overlooked them for a long time because they’re very hard to study and so there’s kind of
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a natural bias to focusing on the structures of the proteins that have structures that are easy
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to study but indeed most proteins especially signaling proteins have a lot of disorder in
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them and it’s important to recognize that and describe structures and signaling Pathways in
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terms of that order and disorder so um this brings us to some of the approaches my labs uses we study
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a number of different proteins involved in synaptic communication and they all share
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certain challenges that make them uh resistant to classical structural biology approaches the the
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details aren’t super critical but we have things like X-ray crystallography and nuclear magnetic resonance and cryoelectron microscopy that help us understand at a really really high resolution the
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shapes and functions of these biological molecules but uh not all proteins are easily explored using
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those classical techniques and signaling proteins are really good examples of that problem they’re
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often composed of multiple different sub-domains regions where the amino acids fold into unique
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shapes with unique functions and they’re all strung together Often by very flexible linkers
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that can be very long and are usually to some degree intrinsically disordered and so these
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proteins move around essentially they don’t sit still for the photo and they uh have resisted
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a lot of the structural characterization that we take for granted in other systems like metabolism
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they’re also famously transient in their interactions meaning they kind of lightly contact
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one another and they fall apart dissociating from one another very easily and that’s a feature not
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a bug uh signaling and communication is all about kind of light interactions assembly communication
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and then quick disassembly when the signal is no longer needed so it’s the reversibility of this
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communication that’s really important so we don’t lock the cell into a particular mode we want to
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be able to reverse this all and easily but that is again a challenge for getting these things
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to kind of assemble hold still so that we can structurally characterize them and how they work
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so we’re we do have a lot to rely on though we have the wealth of work of geneticists
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and cell biologists who have identified the interconnectivity of these different proteins the
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importance of what they do in a cell and at the other end we have the structural biologists who
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have through the decades broken these difficult proteins into little pieces and structurally characterize the pieces and so one of my lab’s motivations in studying how signaling proteins
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work at the protein level is to acknowledge that we do have a lot of great high resolution structural information describing important proteins or these pieces of these proteins and
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our big question is putting those pieces together and describing a higher order architecture of how
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these assemble together and communicate and move with a strong focus on understanding that there
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isn’t a single snapshot to be had here they these are rapidly moving proteins that kind
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of quickly assemble and disassemble and they sort of flex around each other and they Interlock in a big meshwork of other proteins so we need tools that aren’t necessarily going to give us a single
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unitary you know locked in structure but rather tools that can describe an ensemble of structures
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so that the range of motions available to these proteins trying to capture them in action and
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so a kind of newish and exciting approach to this is called integrative structural biology
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and that’s where we kind of try anything we can to get restraints on the structure uh little details
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describing the range of structures available and possible and then we use computers to try to put that together to build a model of how really complex array of proteins can kind of
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Comb together and do their job in a cell and my lab focuses on a collection described in cartoons
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here and I won’t drag you through the details of how all these biophysical things work but the
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higher order or the big picture idea here is that we we do have some reliable models of individual
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pieces of our signaling complexes either through a history of structural biology or through really
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neat machine learning algorithms that can predict with uncanny accuracy what the structures will be
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and then we ask how those pieces fit together and move around each other using different techniques
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um some of them are based on x-ray scattering some of them are based on just basic biochemistry where
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we we try to generate mutants and see what happens to the functions of these individual proteins
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but we also take advantage of a lot of mass spectrometry based tools and this is one of
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the things that makes my lab a little bit odd we do a lot of Mass spectrometry to study proteins
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and that is a technical sounding word and we don’t necessarily need to dissect what a mass
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spectrometer is but I want to acknowledge that mass spectrometry is in its own right a really
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really useful tool for a lot of approaches to studying biology and I want to give credit to
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the people who’ve developed this technology over the years um in general a mass spectrometer does
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a pretty straightforward job it’s able to ionize a molecule and then measure the mass to charge
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ratio and what that comes down to is that we get a really really high Precision Mass measurement
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of individual molecules and with that mass measurement we can identify what molecules
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are in a cell with um great precision and we can survey whole populations of proteins within
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cells and ask how those protein populations change under different conditions either with
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a mutation or maybe in a cancer cell line or in response to different developmental stages
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and so Mass spectrometry has become an actual absolutely foundational technology for proteomics
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which is the field that is responsible for telling us what proteins are doing what and what cells
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uh so that’s uh really important and I know there are some excellent proteomists working
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in the syngap field right now so that I think is what mass spectrometry is most famous for is
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profiling what proteins are doing things in cells and so it’s a little unusual for Mass
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spectrometers to be used to study not the mass of a protein but rather the structure of a protein
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but We’ve Come to embrace Mass spectrometry to do this because it brings in certain advantages
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as long as you apply some tricks to to going Beyond just the Master charge ratio
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and so the the details are kind of technical but there are two versions of this that my lab employs
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one of them is a isotope labeling of proteins that allows us to paint the surface and try to identify
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what’s exposed and what’s Dynamic and contrast that with what’s buried and it’s a it’s called a
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hydrogen deuterium exchange mass spectrometry and I’ll show some examples of that later
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there’s also a cross-linking approaches where we can try to ask which regions of proteins are in communication with each other or which ones are close to one another by kind
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of capturing them with a cross-link a chemical bond between these two pieces so we can then chop
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apart and identify by Mass spectrometry and between these two approaches we can learn a lot about the Dynamics and higher order architecture of different proteins
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so those those are the kind of approaches that my lab uses and we’re currently looking at kinases
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and scaffolds and other things in the postsynaptic density and exploring their structures with mass
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spectrometry and other integrative structural biology approaches but very recently we were
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fortunate to be awarded a a grant to start a pilot study looking at syngap and so
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um my big puck I guess a cautionary note here with the rest of this uh conversation is that we are
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at the very very early stages of This research we have just begun to explore these aspects of
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syngap biology but I uh was really excited to tell you about where we’re going with it and to to get
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your input and hear your questions as well so a bit of background that many of you probably know
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uh better than I do sync app has a really complex role in tuning synaptic plasticity and development
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in neuronal systems it is named for a central domain in it the gtpase activating domain that
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gives it GAP activity but as contemporary research has shown it has many many more roles than that
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it is not simply A protein that regulates G protein signaling and all kind of March
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through some of the aspects of those functions in a bit here but I guess I should reinforce
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just roll on the same page what it means to be a gtpace activating protein
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and that refers to an activity that Tunes a family of signaling proteins that are really
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small and they tend to skate around the membrane and they are often initiators of long elaborate
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signaling Pathways that can often communicate all the way back to the nucleus and control Gene regulation and so Ras and rap are two gtpases that communicate and they do so based on whether or
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not they’re off and bound to a small molecule GDP or on when they’re loaded up with the activating
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GTP and so there are proteins GDP is activating proteins that cause them to hydrolyze their GTP
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and turn themselves off and there are guanine exchange or new guanine nucleotide exchange
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factors that swap out the inactive GDP for GTP and turn them back on again and so uh initially syngap
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was identified as a GAP protein down regulating the communication of these gcpases but as was
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immediately noticed in the first description of the sequence of that big syngap protein it clearly
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had the capacity to do much more it had multiple different domains and a really long apparently
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intrinsically disordered region that could do many more things in the postsynaptic density and so
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that kind of structural and functional complexity is what Drew my lab’s attention to syngap in
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addition to the obvious very serious clinical importance of its function and dysfunction
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and as we were kind of monitoring and following along as researchers were exploring syngap
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function we were really astounded by a couple uh facets of syngap biochemistry and biology
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and so here is just kind of a list of some of the things that caught our attention that made syngap
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a really really strange and fascinating protein uh really something that we kind of wanted to
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explore as well uh the first thing that caught our attention is that syngap is of course one of the
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most abundant proteins in the postsynaptic density and that’s very strange and unusual for a protein
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that’s annotated in at least in terms of its GAP function as being a regulator of another protein
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um hypothetically these are enzymes that can shut off or tune down the activity of gtpses like Ras
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and rap but you don’t really need the GAP to be in huge excess of the gtps is to do that job you
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just need a couple of them you could consider the GAP proteins as kind of management kind of
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dictating what’s happening with a bunch of other squads of employee signaling molecules and you
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you rarely rarely see an excess of the GAP above its substrate uh GTP Aces and so of course uh that
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quickly led to people examining its scaffolding roles like maybe it’s serving a structural role
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in the postsynaptic density like like psd95 and other uh well characterized scaffold in there
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and of course lately there’s been some really astonishing and fascinating developments looking at how the very abundant syngap and its interactions with pst95 scaffolds can cause uh
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kind of hydrogel formation inside the post-actory density or liquid liquid phase transitions
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uh another really curious thing is that there’s been a lot of study about what the target of
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the gtp’s activating activity is in syngap and so there’s been some back and forth about how good it
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is at uh tuning Ras signaling versus rap signaling and what factors are involved in the specificity
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of the signaling pathway and so there’s been some great work from Kennedy and co-workers and others
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looking at how kinases can kind of modulate the activity and the preferences for The GAP activity
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but uh there’s also some really cool new work and I I hope you had a chance to see this I
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think on Monday you can hearing co-workers had a really really cool study where they were able to
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surgically uh kind of ablate the the Gap activity of syngap and showed that there were some really
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really fascinating effects the the primary rules of syngap apparently are not just as a gap protein
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but the dominant role seems to be that scaffolding and that liquid liquid phase transition that it’s
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doing to regulate access to binding sites and to control how many neurotransmitter receptors
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are clustered at the postsynaptic density so this is a really really cool paper that just came out
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as a preprint and uh really really adds uh some complexity to the picture of what syngap is doing
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from uh a structural biology point of view I think you’re probably all used to seeing
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a map of the uh pieces and parts of syngap it’s a really big protein and it’s got that Central gtps
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activating domain that it’s named after but it also has some accessory domains that show
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up in lots of signaling proteins and it has a lot of these in physically disordered linkers
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and so this is just a picture from alpha fold that’s a machine learning algorithm for particular
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structures and I don’t mean to propose this as a legitimate structure here it’s just this is what I
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sees intrinsic disordered regions it just kind of makes this spaghetti wad but it really gives us a
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perspective on how much of the total sequence of syngap is a really really Dynamic wavy
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intrinsically distorted region and this this uh disordered area is very very intimately involved
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in forming those scaffolding interactions and phase transitions in the post-synaptic density
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so my lab is also interested in like these auxiliary domains and what they’re doing
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there’s a domain called a plexton homology domain which was identified mostly by sequence
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conservation it has some of the the kind of sequence features you would expect from a pH domain but it doesn’t apparently fold reliably into a pH domain and there’s
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still a lot of questions about what it does maybe ancestrally it came from a
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sequence that folded into a pH domain but maybe a diverged over evolutionary time it also has a C2 domain and C2 domains are famous for binding and responding to calcium which is
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intriguing because there’s a lot of synaptic signaling involved in calcium flux and there was
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kind of Interest initially in considering that syngap might be a calcium responsive but while
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structural predictions suggest that the C2 domain does have a pretty classic or C2 domain fold it
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doesn’t have the actual residues that would be responsible for binding calcium so it seems to not respond to calcium and there’s some really cool structural work that suggests that the C2
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domain is involved in selecting or nestling into the GTP Aces that sing out might tune activity of
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so yeah this is just a whole list a laundry list of things that remain unclear about the
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structure and function of syngap I think it’s been pointed out before that despite many of
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these regions not having obvious structure or function they’re still quite conserved they’re
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surprisingly conserved in manuals and they’re even conserved across birds and and reptiles and
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things like that so there is appears to be some long-term selective pressure for maintaining these
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unusual syngap features and this again urges us to explore how they work at a higher level of detail
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so my lab and many other labs are interested in these big overall mechanistic questions for one
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you know what is the dynamic architecture of this whole syngap molecule lately there’s been
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astounding work showing that it can trimerize and form these condensates and we’d like to
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explore how the rest of the domains of syngap behave in the context of those condensates
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there’s also long-standing interest in how the auxiliary domains as well as post-translational modification influence its enzymatic activity and its scaffolding activity
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and I also think it’s important to think about syngap as being a protein that is right next to the membrane because the gtps is that it does whose activity it does tune
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these are peripheral membrane proteins kind of skating along the edge of the synapse membrane
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so I need to consider how the membrane might be influencing its activity
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and of course the exploration of the higher order scaffolding interactions is very key
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thinking about uh the competition between those interactions as well it’s probably not just a
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static web of interactions between syngaptrimers and psd95 but rather exchanges of these binding
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sites that are allowing changes in the number of receptors that are in the membrane and wonderful
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work is being done in that field as well and of course my lab is uh interested in a lot of
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post-translational modifications uh the kind of molecular flags that change and shift how
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signaling proteins communicate with one another and syngap is indeed subject to phosphorylation
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one example of that plus violation can tune its activity which usually comes about by tuning its
34:06
structure or allowing it to interact with new things so we’d like to explore that some too
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so uh what we had proposed to do was look at a lot of these protein Dynamics and structural questions
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using our kind of audible Mass spectrometry approaches and uh these are the two I mentioned
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before and uh it’s I guess useful to ask you know why why are we employing Mass spectrometry
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to do this and the reason is because uh the mass spectrometry technology itself is really really
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easy to apply to humongous proteins and to protein complexes and that can be a challenge for a lot of
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the other more traditional structural biology approaches you can also make these structural measurements in the context of membranes which are themselves very fluid and challenging for
35:02
structural biology we can build up from the bottom up from purifying multiple different
35:08
proteins and putting them together and see how they assemble like Legos and we can kind of probe those bigger structures as well using the same techniques and we also
35:18
don’t need these proteins to sit still they can continue to flex and twirl and move around each
35:23
other and these Mass spectrometry techniques are able to capture the changes and Dynamics
35:30
associated with the linkers and intrinsically disordered regions of signaling proteins
35:36
so um we’re just at the very beginning of this we just got the the research Grant uh two months ago
35:43
but we are excited to apply these approaches to syngap and we have a lot of different questions
35:50
that we’d like to look at in our remaining time I kind of want to give you just a
35:57
slight glimpse of what that would look like um we won’t go into this too much
36:03
here but I wanted to show you an example of hydrogen deuterium exchange mass spec
36:08
this is considered a footprinting approach where we can use a mass spectrometry readout to examine
36:15
the dynamic surfaces of signaling proteins and look at how the surfaces interlock and how
36:22
they move and how they change when they’re modified with phosphorylation for example
36:28
and so it involves taking a feature that’s Universal in proteins proteins are made up of
36:34
individual amino acids like this one and this one that are linked together in peptide bonds
36:40
and every one of those peptide bonds and chemically exchange with the water solvent
36:46
and so this is happening constantly all of your proteins and all of your cells are exchanging their atoms specifically the hydrogen atoms with the water in the cell but we never really notice
36:57
it because it’s just a hydrogen exchange for hydrogen but we can actually measure that rate
37:02
of exchange how quickly that happens by using isotopically labeled water and this is just a heavy isotope that’s not Radioactive It’s just got one extra Neutron but that one extra
37:13
Neutron is something you can easily measure with mass spectrometry and so we can profile where
37:19
um where in the protein it readily exchanges what the water surrounding it and that can tell us what’s on the surface and what’s buried in interactions and what’s moving really easily
37:30
so here’s kind of a cartoon version of that this is kind of an example protein and if you dip this protein into isotope labeled water you can see exchange all around
37:39
the surfaces wherever it’s kind of moving and dynamic and we can profile proteins and learn
37:46
a lot about their structures using the rates of those exchanges and some of the things we
37:51
can learn about are kind of substructures that we call secondary structure we can learn about
37:56
how quickly different parts of the protein are moving we can map how proteins interlock
38:02
and recognize each other which is important for communication and we can learn things about the
38:08
subtle local chemical environments which would be perturbed by a phosphorylation for example
38:14
and at the end of the day if you skip past all of the Mass Spectrum detail what we get is uh data
38:23
that we can color code and this is popular in my lab my students like to look at these things in
38:28
color code terms um it’s much more easier and much easier than just looking at tables and numbers
38:34
so um this is kind of just a generic example of some data that we took these are two different
38:41
kinases that we were exploring and you know these are the structures of them and we kind
38:46
of knew the static structure from old structural biology approaches but we wanted to ask uh what
38:53
parts were Dynamic like we can get a more action shot of these things and so we did some hydrogen
39:00
deuterium exchange mass spectrometry and we could kind of map over time where exchange happened and
39:06
we color coded that going from low exchange and blue to high exchange or high Dynamics in red and
39:13
you can kind of see the periphery and the edges and the loops exchange and get redder over time
39:18
and that’s the kind of data that we can extract and use to build models of signaling function
39:25
so um this is just a cartoon to recognize that these are comparative experiments we can look at
39:34
a protein in one state and look at it in another state and compare the Dynamics and so we might
39:41
look at a protein that’s not bound to a partner like a scaffold and one that is bound and we can
39:46
look at how the Dynamics change and So currently we’re we’ve already started that looking at
39:52
syngap and how its exchange rates tell us about interactions And We Begin by looking at those
39:58
auxiliary domains if we compare a syngap with its C2 domain cut off or the holson Gap we can examine
40:06
how it interacts with the G protein and indeed we can show the regions color encoded in blue here
40:12
that uh kind of are impacted by The Binding and Association of one of those G protein substrates
40:20
and interestingly even though we think of the Gap domain as being the heart of that activity we see
40:26
most of the changes in the C2 domain which was predicted by the the structural biology group that
40:33
initially co-crystallized these together so we’re also looking at how membrane interactions would
40:41
tune the recognition of syngap with its substrates and that’s important because Ras and rap are both
40:48
gtpases that anchor to the membrane and so the membrane often has a really important role in that
40:55
so um I think just give you some uh quick uh examples of what we’re moving towards I
41:04
won’t go into the details of this kinase activity for time’s sake but we were doing very parallel
41:10
experiments looking at a kinase involved in synaptics plasticity and we were able to look
41:17
at how its different domains contact each other in the same way and again we love color coding
41:23
so we were able to map how a regulatory domain interacted with the signaling domain and we could
41:29
see an imprint left by their interactions where the exchange rates were slowed and we color code
41:34
those in blue and we were able to build a model of that interaction and then look at how things
41:40
like phosphorylation change that interaction and so we see these blue kind of uh stabilized
41:46
regions of the higher order protein totally reverse themselves when you phosphorylate and now they swing into extra Dynamics and more readily exchanged and we could go
41:56
zooming in real detailed and learn about catalytic mechanisms and signaling mechanisms through that
42:01
and so I wanted to make sure we I had a time to to meet some of you and hear some thoughts
42:08
and questions so uh with that I kind of want to again uh acknowledge my team um Quinn and Sarah
42:15
are the two that uh began this project building up our syngap infrastructure and the ones who
42:23
uh interactions with GTP Aces and profiling the Dynamics of that tail region
42:30
and I want to acknowledge the future colleagues working on this uh two grad
42:37
students and a new team of undergrads and we’re recruiting a new person just at the
42:42
end of this month and the funding source that we have to support that research most recently
42:49
the syngap research comes from the NIH NIMH so I would welcome any any questions or thoughts
43:02
yep let me grab a scat but
43:07
um if anybody wants to answer ask any questions you can type them in the Q a or on Facebook
43:12
in the comment section and then Janie Reed is here too she I know has some questions I’m sure
43:21
um so go ahead hi Janie hi Eric thank you so much this is so incredible and Lauren you can see the
43:31
um questions right yeah do you see any no I just don’t see any yet but yeah I can
43:38
but yeah it’s gonna be in the chatting q a it opens yeah
43:44
so yeah I’ll let you know oh does Mike want to say something before he goes he’s needed
43:51
uh I I am going to run out I have to run children around unfortunately but I was going to say we
43:56
have a pretty intimate group although I’m sure this will be watched on on video many times
44:02
we have some esteemed researchers in the um in the attendee section so if any of our
44:08
other syngap scientists want to ask questions J.R and Lauren you might just want to promote them to be able to speak and I just want to express my thanks Dr underbocky and regrets
44:19
that I won’t be here for the rest of this all right have a good day guys thank you
44:27
yeah so this is JR this is Lauren please um promote any of the scientists that I
44:33
think maybe Dr wilsey’s here I don’t know I can’t actually see his end so yeah so
44:43
um I think my first thing I want to do is just give you so much thanks this is incredible like
44:49
absolutely incredible like exactly the kind of structural information we’re gonna want and need
44:56
I think one thing that’s really interesting about syngap uh related sync F1 related and intellectual
45:03
disability is that it’s really considered a have low insufficiency and so I’m I I can’t wait to to
45:15
see all the all the understanding of what syngap’s doing but I also kind of am interested in how
45:22
you know just having half of it around makes a problem and then in addition to that to the Miss
45:27
sense how oda’s having one amino acid change out of this whole thing make make it look like
45:37
it seems like maybe it’s looking like a haplo insufficiency so um or or maybe maybe it’s not
45:43
a house maybe the maybe those aren’t hablo insufficiencies and is will there be a way that you can tell between a null a high some more hypermorph neomorph and a dominant negative which
45:55
are kind of the maybe there’s more categories than that but those are kind of like the five big ones
46:00
that I think about yeah that’s a excellent observation and a really profound question
46:06
I think is being addressed community-wide our role in that is only a fraction of it
46:14
I do think the the last 10 years of work that have shown that you know obviously haplo insufficiency
46:22
dominates this and that isn’t the kind of thing we would expect from maybe an enzyme that was
46:30
just controlling gtpa signaling it’s clearly something broader than that because there is such a sensitivity to the population of Stoichiometry within the context of the postsynaptic density
46:40
and I think the the more recent work showing scaffolding roles phase Transitions and that neat
46:46
slot idea controlling how many neurotransmitter receptors are available to Cluster at the PSD
46:52
as opposed to diffusing through the membrane I think those are really revelatory in terms of
47:00
why you see this range of consequence when there is haploinsufficiency there’s also the
47:07
really interesting notion of point mutation single residue mutations that can cause
47:16
very big effects which is on the country not what you might expect if this was doing
47:23
just a scaffolding role and so um it seems to be an interplay of a couple different
47:28
things and maybe they work at different time scales into different stages of development certainly my lab alone is not uh sufficient to answer that it’ll definitely be a community-wide
47:39
effort to explore those things but in addition to what the rest of the community is doing Labs like
47:45
mine that can look at a structural level are very very interested in this looking at how individual
47:50
point mutations can change the global Dynamics or local Dynamics or the functions of the Gap domain
47:59
is a really cool detail in that new preprint too about personalized genomics identifying
48:09
um actual mutations and gapamines that don’t appear to have a function even though you would think that they would have a severe impact on the catalytic function and so uh it’s research like
48:19
that that’s able to look at the role of the scaffolding the role of the face Transitions
48:26
and compare that to whatever their roles there are for controlling uh small gtps signaling too
48:32
and so um what we’re looking at now in my lab are we’re very excited to look at missense mutations
48:38
that are known to have actual impacts on on people and we’re also interested in exploring
48:46
the scaffolding interactions which is something that we’re we’re eager to do it’s especially with
48:52
cross-linking there are known differences in how scaffolding interactions change especially
48:57
when phosphorylated by a chemk2 and we can see that sort of interaction Network that causes
49:03
the phase transitions can be tuned uh based on post-translational modification and that’s
49:09
really important it looks very much like all the literature is pointing to that this is not just some sort of static stable structural Force it’s a structuring or a scaffolding that is responsive
49:21
to patterns of neurotransmission sorry that was kind of a Meandering answer but I I agree
49:36
I guess I I did talk to you before so I do know that you’re going to look at some sense changes
49:41
and that’s very very exciting and there’s so many aspects of where your work go that it’ll be
49:48
interesting to see what ends up being what ends up jumping out first is what you want to look at more
49:54
um I want to have Dr Helen mulsey talk and so she has a question in the in the Q a and I’ll just
50:05
read it out loud so that maybe you and she says might have missed it how are you thinking about cell types specificity of isoforms your mass spec approach seems perfect to understand isoforms too
50:18
oh that’s a really good question I think there’s two levels of that one would be I’ll
50:25
give credit to the proteomistus who I know are actively working on this that are are definitely
50:31
um people who are looking at different cell types to different stages of development and using the power of Mass spectrometry to identify the abundance and uh I’m not 100 sure but I I hope
50:44
I expect they’re probably also looking at subcellular localization as well like the sort of patterns of movements of this in gap and um absolutely the the role of isoforms and
50:55
different expression patterns of those isoforms is really really critical to this and um I I totally
51:03
agree that uh cell type differences are probably very profound um as well as developmental stages
51:11
um on the other side my lab is primarily focused on the fine-grained protein details so we we are
51:19
absolutely interested in isoform differences um we we can readily clone different isoforms and we can
51:26
sort of produce the individual protein but that that’s our take is usually building up from the
51:32
sort of molecular level uh ground up versus top down and that’s the kind of perspective that that
51:40
we’re supplying but definitely looking forward to both exploring isotope or isoform differences
51:46
as well as the impacts of known mutations on the Dynamics and interactions and Scaffolding
52:02
okay well Dr Wilson do you have any follow-up questions on that no that was great I think it’s really exciting you know when I think about isoforms and I look
52:11
at sort of the recent work on all the C terminal ones I think they’re really interesting right and I think part of understanding how that affects the structure of the protein and then also its
52:21
interactors I think could be super cool so it’s great that you’re going to take a look
52:26
absolutely and I appreciate those thoughts and questions I I share that interest and it’s
52:32
um going to be really really fascinating to see as different groups focusing at those different
52:38
scales of complexity come together uh and and reveal how those changes impact that
52:46
Global PSD organization I think that’s just the remarkable thing especially with some of these
52:54
um face separation studies there’s syngap is a very Central player in that density organization
53:08
okay can I ask a pretty basic question about structure so there’s that really long coiled
53:17
but that really long coil and I’ve heard that referred to as maybe the quilled coil domain it’s near the sea terminal and I’ve heard that that can form a trimer do we how much is actually known
53:30
about whether it does form a trimer in Vivo if um if there are different isoforms that primerize
53:38
that the same you know in the same timer and if the you know if you have a missense mutation how
53:45
how much might that how much my Oneness sense with two wild type or two minnesotans with one
53:52
wild type how much might that change function is is that anything you can start to get at here or
53:57
do you mostly look at single uh single proteins absolutely uh that’s a really good question and
54:06
something we are actively thinking about in the exact same way the the original Revelation that this good form of trimer was uh really fun and exciting the a lot of the work was done with a
54:18
piece of that tale of syngap just for practical reasons it’s very hard to make the whole protein
54:24
and to study those higher order interactions it looks like that piece looks really stable
54:31
if you purify that uh portion a syngap that research was really strong and it looks like
54:38
that timer really can form and especially in the purified setting and it does seem to form that
54:46
then higher order lattice work by interacting with PTZ domains or pst95 the in Vivo part though is is
54:53
the real fascinating question um the postsynaptic density is full of things that compete for
55:00
interactions with psd95 and the PTZ domains and so it’ll be interesting to ask questions of how
55:09
Dynamic this is and I think it is clear that it is highly Dynamic um that that new preprint had some
55:16
really fascinating stuff looking at um competing phase Transitions and sort of phases within phases
55:24
um the there’s also like what happens when you phosphorylate in that region does that loosen
55:33
or tighten up those trimerization interactions or the interactions with other scaffolds and that is
55:39
something that we’re exploring as well it’s pretty straightforward for us to do that in a in a really
55:49
reductionist setting uh to explore like primer is interacting with other scaffolds and looking
55:56
at the consequences of phosphorylation but I also think it’ll be really important to study that in
56:01
a Cell based system which is a little outside of our wheelhouse but we I’m confident that there are
56:06
other labs working on that too and in that cell based setting we would you would bring in the kind
56:12
of context of the competing interactions like how how does this kind of Dynamic competition of these
56:18
same limited binding sites work instead of just looking at it with one or two purified proteins
56:24
or or even three or four purified proteins the global PST is a much more complicated place
56:35
I want to think you’re asking about mutations as well that
56:40
that is also something we’re thinking about and I’m I’m intrigued by um
56:47
from our uh broader experience with other signaling proteins that primer paper really
56:53
really did seem like it was quite a stable interaction and um
56:59
it probably isn’t quite that locked in in the cell and there’s probably again aspects of having competing interactions that are involved in that and I think if that were
57:10
a little held together more Loosely which would be more typical of other signaling proteins than
57:17
you could easily see that being very vulnerable to perturbations like the sense mutations and I
57:24
think that’s also reflected in the sequence conservation of this through other organisms
57:30
it is highly unusual to have this degree of conservation um in regions that could otherwise
57:36
just be there for the flexibility it does seem that that conservation hints of hints at specific
57:42
interactions with other proteins that would then uh have real consequences if they were mutated
58:00
yeah any other thoughts
58:10
I like check Facebook again one more time real quick sure well there’s a lot I again
58:17
wanted to thank you for the opportunity to interact with this community I’m really really you know what you are doing and it’s very very exciting to get a chance to share our
58:28
at least uh the beginnings of our work in this area thank you so much as well yeah this is great
58:35
well I don’t see any on here uh Jr do you have anything else or
58:41
no I was scattering away on mute but I um during that level but okay well I I think I think that’s
58:50
I think that’s great I mean I I think this is I I think the main thing is on you know we have a a
58:58
grant cycle on you know for for September 1st is when we accept grants for our uh on a six-month
59:07
cycle September 1st and I guess February 1st so if if you want to write a grant to have a postdoc
59:14
help you with this please do be this is exactly the kind of work that we’ve been hoping for we
59:21
just didn’t know how to say it because we didn’t know any of this but yeah structural understanding
59:26
the structure of syngap and understanding how uh single nucleotide changes could in in you know
59:35
all over the all over the protein how they could potentially be affecting things but I’m so glad
59:42
to hear it that I’ve heard that the domain of unknown function but you’re kind of calling it this unstructured you know domain of unstructured event structure is really really interesting so
59:54
I’m I’m really excited to continue to be in touch with you I’m really glad that you told us about this at the beginning and um we’re all rooting for you and your team to get lots of clean data
1:00:06
thank you so much I was so happy that you all reached out and it’s great to make these
1:00:12
connections thank you okay well how everybody have a good day thank you so much bye bye