Wednesday, January 22, 2020

Secret Science Club Post-Lecture Recap: Cellular Switches

Last night, I headed down to the beautiful Bell House, in the Gowanus section of Brooklyn, for this month's Secret Science Club lecture, featuring structural biologist Dr Kevin Gardner, Einstein Professor of Chemistry & Biochemistry at City College of New York Director of the Structural Biology Initiative at CUNY’s Advanced Science Research Center. Dr Gardner titled his lecture Harnessing Nature's Switches: Discovering New Biotech Tools and Drug Targets.

Dr Gardner began the lecture with a question- how do cells 'listen' to the world around them and act on that information? Cells have to respond to outside stimuli second to second, minute to minute. Figuring out these mechanisms is important to biomedical research. Cells have the ability to sense oxygen levels in order to respond to conditions of hypoxia. Plant cells have to sense light levels so they can discontinue photosynthesis after sundown. Fish have the ability to sense pollutants in water, with the sensitivity callibrated to pollution levels. These cellular 'switches' are variants of the same basic process, which involves small molecules, such as the protein rhodopsin (derived from vitamin A). The switches get proteins to change shape in response to stimuli.

Dr Gardner told us the mantra of structural biology: Structure Determines Function. Biology can be likened to the opposite of architecture... in biology, structure determines function, while in architecture, function determines structure. An architect tasked to design a concert hall will base the structure on the acoustic needs of performances. With biologists, evolution is the 'client'. Biology has produced some good structures, but a lot of okay structures.

In the 1950s, the structure of DNA was determined, creating the models involved a lot of chemistry. Around the same time, the role of proteins such as myoglobin and hemoglobin was being studied. New insights into disease, such as the role of a single point mutation in sickle-cell anemia, were being made. Dr Gardner then took a moment to recognize the role of graduate students, who do most of the work in scientific research.

Dr Gardner then stated the multi-disciplinary nature of structural biology- structural biologists answer biology's questions, harness chemistry's intuition, and use physics' tools. In order to view the small molecules used in cellular switches, electron microscopes are needed. A typical human cell is ten to thirty microns in diameter. Images of cells must be 'blown up' to allow researchers to 'look around'. Using the example of a myoglobin molecule, Dr Gardner noted that, if a cell were blown up to the size of One World Trade Center, a myoglobin molecule inside it would be an inch in diameter.

Dr Gardner then described the various instruments of his trade (the 'tools of physics') in dramatic terms. He joked that they constituted 'science built on a dare'. Cryoelectron microscopes use extremely low temperatures to reduce damage to biological specimens, allowing 3D images to be obtained (Dr Gardner quipped, "Don't mess around with electron guns"). Nuclear magnetic spectroscopy (Dr Gardner noted wryly, "Anything nuclear is good") involves using a large 'Thermos' with liquid nitrogen and liquid helium chambers wrapped with thousands of feet of superconductor cables, and utilizes a localized magnetic field millions of times more powerful than that of the Earth to obtain images of proteins- Dr Gardner described it as 'an atomic MRI'. A more 'sane' method of imaging molecules is X-ray crystallography, in which molecules in rows in a crystal have x-rays shone through them- the x-ray being intense enough to set air on fire. Again, Dr Gardner joked that this was 'science built on a dare'.

After this overview of imaging methodology, Dr Gardner got to the core subject of his lecture, beginning with an introduction to G Protein-Coupled Receptors, which are found in cell membranes and trigger cellular responses to outside molecules. GRCRs are the target of two-thirds of all pharmaceuticals- the drugs hijack GPCRs to result in a clinical outcome. Rhodopsin is a GPCR found in the human retina. Protein domains are pieces of protein, and Per-Arnt-Sim (PAS) domains are the protein pieces that act as molecular sensors. Three PAS domains were identified in 1991, and there are currently about forty-thousand known PAS domains.

Light-oxygen-voltage-sensing (LOV)domains are PAS domains which detect blue light, they can be turned on or off by shining blue light on them. LOV domains contain a derivative of riboflavin. They convert blue light into biochemical signals which are also dependent on other factors. One of the processes controlled by LOV factors is phototropism, the movement of plants toward light sources. If the PAS domain regulating the sensing of blue light can be knocked out, no phototropism occurs. In plants, the 2LOV domain activates a kinase which triggers the response. In darkness, the flavins are bound covalently, blue light creates new bonds to turn on the kinase. After checking with Dorian and Margaret that the Bell House crowd was over 21, Dr Gardner declared this process 'batshit crazy'. Blue light synchronizes the switches, which shut down after sundown. Rhodopsin in animal eyes shuts down images rapidly, so they don't persist, but plants don't need quick reaction times, so the light-detection-and-reaction process is slower than that of animals... Dr Gardner describe it as a 'good enough' evolutionary solution. Laser equipped NMR spectrometers can be used to study phototropics such as LOV2, which covert changes in light into changes in activity.

Besides studying PAS domains in the lab, biologists sequence DNA from a variety of organisms in order to identify proteins. Dr Gardner cited the 1KP Project, which sequenced the genomes of 1,100 plant species. This is being followed up with the even more expansive 10KP Project. Dr Gardner described a 'bioinformatics pipeline' from the field to the lab, and quipped that 'Nature's been busy'. He also stated another scientific mantra: MODELS GOOD, DATA BETTER.

He took a brief moment to express some envy of the scientists working in the field... he's a recreational diver, but the oceanic data has been outsourced to other researchers. He has, though, done field work in a local swamp. Using this as a transition, he then brought up the subject of the bacterium Erythrobacter litoralis, the genome of which was sequenced from a specimen collected in the Sargasso Sea. This bacterium is an opportunist, it can use photosynthesis to provide energy. Dr Gardner explained the significance of blue light- it penetrates deeper into seawater than other wavelengths. Erythrobacter litoralis has sensors which check light levels, which are dependent on both time and depth. A light-activated binding protein (EL222) was discovered in Erythrobacter litoralis, light 'flips the switch' to turn genes on. Other blue light sensing proteins are also known. Dr Gardner posed the question, are these 'bacteria only' tricks, or do eukaryotes use them? LOV PAS domains have been found in fungi such as brewers' yeast. Detection methods used to examine to action of photosensory and binding domains, and the changes in shape caused by exposure to light are not friendly to living organisms. To study these proteins, researches set beacons in the domains, flash them, and measure the changes. Because the LOV domains respond to blue light, these researches work, like Roxanne, under red light.

Dr Gardner told us to keep our eyes on optogenetics for an upcoming Nobel Prize. Place genes in organisms and use novel ways to illuminate them. Dr Gardner showed a video (which I haven't been able to locate yet) of a rat which had had an algal gene inserted into it using a virus, and a fiber optic cable inserted into its brain so neurons could be turned on or off using lasers, resulting in behavioral changes, in this case, feeding. After this look at one class of algal photoreceptors, researchers can go on to other photoreceptors, of which thirteen classes are known, detecting light from the ultraviolet to the infrared. Putting bacterial receptors into eukaryotes poses some issues- the receptors have to deal with the presence of a cell nucleus and 'figure out' the mechanisms of the eukaryotic cell. The EL222 protein has been turned into a research tool, inserted into organisms such as mice, yeast, and zebrafish. The more the gene expression in an organism, the more the response to light. The proteins which 'see light' in plants bond to other proteins and change their shape have been turned into biotech tools.

After this introduction to light sensors, Dr Gardner shifted to the subject of hypoxia sensors, the subject of the 2019 Nobel Prize in Physiology and Medicine. Hypoxia-inducible factor 1-alpha (HIF1A) is a PAS domain made in cells. HIFA1 is destroyed in real-time under normal conditions. If not enough oxygen is present, HIFA1 is not destroyed, which set up a response to produce more ATP (the fuel of the cell), and animals produce more red blood cells are reroute blood circulation to oxygen-starved body parts. HIFA1 can be used to treat anemia or blood circulation problems, but it also has the potential to be used for 'doping' by endurance athletes. Dr Gardner ruefully noted that speed walkers had beaten cyclists to the punch when it came to doping using a drug which inhibits HIFA1 destruction. Genes are turned on when they are needed. The genetic factors which produce growth can promote unregulated growth, cancer. If the system is broken, it is possible that shutting down HIFA1 production in cells can inhibit cancer. The structure of HIFA1 is similar to the structure of phototropic PAS domains.

Dr Gardner finished up with an exploration of biomedical research. One ongoing project is the building of 'libraries' of chemicals which could potentially be the 'grandfathers' of medicines. Fragment-based drug discovery involves observing where various chemicals bond, to which proteins. Currently, two-hundred thousand compounds are being testing for their potential as medicines. Do the compounds bond? Do the compounds do something functional? So far, eighty-five compounds merit further testing. He himself was involved with a small company, Peloton Therapeutics, which evaluated an HIF inhibitor (PT399) which might be used to treat kidney cancer, and is now in clinical trials. Peloton Therapeutics was bought by Merck. Dr Gardner noted that while plants and humans have different biology, they have similar regulation- plant work was what got us to bacterial work which got us to work on humans. He also reminded us that the biologists couldn't have gotten to this stage without physics and chemistry. Research is expensive, but drug costs are high for non-academic reasons... small companies take on the drug discovery risks and get bought out by pharmaceutical giants when they become established. Dr Gardner ended the lecture by thanking all of his grad students, and suggested that the audience buy these good people a drink in gratitude.

The lecture was followed by a brief Q&A session. Some Bastard in the audience asked the good doctor if current models proposed a single origin for blue light sensing protein domains, or if they evolved multiple times in different lineages, was it divergent or convergent evolution? Dr Gardner stated that there is good genetic evidence that blue light sensors are all derived from a common ancestor. Another questioner asked about circadian rhythms- even fungi such as bread mold have cues for timing. Light and feeding tend to correlate. Another question involved (I'd love to know what inspired this) the role that oxygen sensors might play in the incidence of obesity in high-altitude populations. Dr Gardner noted that populations in the Himalayas and Andes have evolved various HIF-related ways (more than one) to solve altitude issues. In the Himalayas, specifically Tibet, there are mechanisms which turn down HIF domains to prevent overly thickening the blood with red blood cells, but these have not evolved in populations in the Andes.

Dr Gardner delivered a great lecture, hitting the audience with a lot of technical terms but remaining accessible. I, personally, found the tying together of blue light sensors and hypoxia sensors very enlightening... it all comes down to proteins, and I find the use of similar 'toolkits' to solve different problems to be elegant. He threw in enough humor to keep the lecture from bogging down in the details. It helped, though, to be familiar with terms such as 'kinase' (this is where Secret Science Synergy comes in-the more lectures you go to, the better each one gets. Kudos to Dr Gardner, Margaret and Dorian, and the staff of the beautiful Bell House.

I've been poking around for videos of the good doctor lecturing, but there are a lot of Kevin Gardners, musicians AND biologists, out there. Here is the good doctor speaking at a CUNY open house about his research, starting at the twenty-seven minute mark:

Grab yourself a beverage, check it out, and soak in that SCIENCE!


Kdus232425 said...

Thanks for the posting. Science advances! How to get in the game?
Invested almost 30 years ago in this now defunct shell company controlled by Carl Icahn which mastered g-protein receptor programming of yeast cell lines. Now with Crspr-cas9 and more than 90,000 individual genome samples courtesy of DoD personnel under control of Patrick Soon-Shiong (Abraxane fame) at the John Murtha Breast Cancer Center in Windber Pa and the new Great Game is on.

Big Bad Bald Bastard said...

How to get in the game? Start off with a lot of money, I'm sorry to say.

Kdus232425 said...

Hahaha! I could tell you understood my question to be rhetorical notwithstanding the hijacking of research usually via lawfare.
I'm no longer the 29yo self I was in 1991 either. Although, I still hold those 200 KDUS shares in case Carl still wants to deal. Funny too how the vernacular changes even in science as we rarely here the term 'monoclonal antibody' anymore.

Big Bad Bald Bastard said...

1991... the world was so different then!

Kdus232425 said...

Not so different that a chemist and new doctor recognized the enormous importance of G-protein receptor coupling biochemistry. Ten years before that, this same chemist not doctor yet, proposed to piggyback a colleague's bench research on GaAs materials for semiconductor chip making. The proposal was rejected by the navy university and the rest, we shall say, is the history of cellphone development.
Ever heard of acquisitive projective Identification?