My Journey into Science: Participation to Passion

I’ve always loved science. I even love the word. Those aptitude tests we all take in school also knew I loved the natural sciences. In high school, Biology came easy for me, and I took it all in (thank you, Mrs. Hill). It continued into my community college experience in Biology for Majors (thanks, Dr. Fleming). Then came my time at a big university where I even retook biology classes as electives because I loved the subject matter (thanks, Dr. Schwartz). As a pre-med, I was expected to take a lot of life science course work, and I gladly did. Then came Biochem, the upper-level weed-out class. I was young and immature so, needless to say, I did not take it too seriously. It showed in my grade.

At this time, science classes were still a set of facts needed to be memorized for the upcoming exam. No way to approach a life-long passion. The life sciences came easy and all the textbooks over the years made it seem easy. The textbooks laid out the facts in front of me; plain and simple. If I had a question, the answer was right there (after a gaze at the index pages). Biochem became my nemesis. I loved it, but it did not return the favor.

Then, it hit me (thanks, Dr. Koontz). These are not static facts. Everything is connected creating a mesh of life sustaining processes. The revelations did not stop there, however. I was fortunate enough to win a summer internship at the Oak Ridge National Laboratory working on hydrogen evolution from spinach photosystem I. This was my first lab experience…ever. The notion of science being easy and quick went out the window soon after beginning. Science, real science, was hard and time-consuming. Science was frustration and troubleshooting, and I loved it.

The only way to truly understand the essence of what science is and how discoveries are made is by performing the work necessary to obtain new knowledge. The discovery timeline needs to be emphasized in science classrooms. Discovery and innovation are not immediate. Hard work and perseverance are vital. I would love to start a page at Sci of Relief entitled Science Timelines. The truth is much more astounding than the myth of science being a series of Eureka! moments.

Bacterial Chemotaxis and Human Memory: Pete and Repeat

Many do not place ‘bacteria’ and ‘memory’ in the same sentence. Normal human perception does not connect the two concepts. However, Mother Nature seems to have a more profound perception. The past 50 years or so of scientific investigation has shown how our uniqueness as humans is actually commonplace across all forms of life on Earth. Case in point, how closely associated molecular memory is between bacteria and human.

Bacteria use adaptation to signals as memory

Swimming bacteria do not move randomly in their environment. This behavior would be futile and counterproductive. Instead, bacteria are constantly monitoring their environment in search of food and poisons. Moving towards the former and away from the latter. This observation was first published in the late 19th century. Bacteria, like the famous and infamous E. coli, use molecular antennae to receive these important ‘signals’ as the basis in the decision of which direction to swim. What if the bacteria find a great place to reside with lots of food but still need to receive signals to ensure they remain there? The antennae have sections that can be modified easily and reversibly. These modifications, in the form of methylation, alter the sensitivity of the antenna protein to subsequent signals. Methylation allows these antennae not to receive the number of absolute signals but relative signals. In other words, the antenna protein through fine-tuned methylation detects changes in the number of signals now versus some time in the past. This is the basis of molecular memory.

These antennae are proteins called methyl-accepting chemotaxis proteins, or MCPs. MCPs accept methyl groups from the essential cofactor S-adenosylmethionine (aka SAM or AdoMet). AdoMet is essential to both prokaryotes and eukaryotes like humans. The methyl groups are added by a protein called CheR (pronounced ‘key R’) which transfers the methyl from AdoMet to very specific amino acid side groups of glutamate. The process, called O-methylation adds the methyl group to the single-bonded oxygen on the carboxyl.

O-methylation reaction
O-methylation reaction. Courtesy of www.brenda-enzymes.org.

The length of a bacterium’s molecular memory is very short in comparison to how we perceive memory at only a few seconds. But, to bacteria it is long enough to successfully navigate the environment with similar precision when concentrations of food or poison vary (up to several orders of magnitude, or ~1000x).

Does the basis of molecular memory in humans mimic bacteria?

Eukaryotes, including humans, use a very similar mechanism in signal transduction to bacteria. Phosphorylation (transferring a phosphate group from ATP or GTP to a protein amino acid) is the basis of all signal transduction and cell regulation. Bacteria use histidine kinases and response regulators, as do plants to some degree. However, the majority of regulation through signal transduction in eukaryotes is through two types of proteins, RAS proteins and the heterotrimeric G-proteins. G-proteins interact with membrane receptors that regulate their activity. What determines which surface receptors G-proteins interact with? Isoprenylcysteine methyltransferase, or ICMT, is one of two methyltransferases that regulate signal transduction activity. ICMT is a membrane protein that uses AdoMet to add methyl groups to isoprenylcysteine, a post-translationally modified cysteine residue on both heterotrimeric and RAS-related G proteins. Methylation regulates which receptors the G-proteins interact with, thus playing a major role in connecting the initial signal to downstream regulatory pathways. The carboxyl methylation essentially modulates G-protein signalling globally.

G-protein carboxyl methylation is regulated by GPCR signaling and, as seen above, GPCR signaling is regulated by G-protein carboxyl methylation. This feedback/feed forward loop could be seen as a form of molecular memory stored in methylation patterns. Within the brain, ICMT activity is almost exclusively found in the region controlling coordination of movement. Thus, methylation could be used to modulate certain neuronal signaling pathways which result in learned patterns of sensory-motor skills.

The only other major methyltransferase is from a protein known as PPMT. PPMT interacts with a major enzyme in signal termination, the protein phosphatase PP2A. PPMT adds methyl groups to the backbone carboxyl of a specific leucine in PP2A. This carboxyl methylation helps determine which B subunit PP2A interacts with and where in the cell PP2A can be found. PPMT structurally resembles CheR in bacterial memory. Moreover, the enzyme that removes the methyl group from PP2A, PME, structurally resembles the bacterial enzyme that removes methyls from MCPs, CheB.

PP2A is one of the major regulators of pathway coordination to maintain synaptic plasticity in the brain. Interestingly, methylation defects and PP2A-PME complexes are suggested to play a role in the cause of Alzheimer’s Disease and memory loss. Methylation defects leading to defective phosphatase activity of PP2A leads to accumulation of a phosphorylated subunit of the structural protein microtubule. In this phosphorylated form, the filaments used to keep axons structurally sound collapse and lead to loss of normal synapses. Therefore, molecular memory in the form of methylation plays a vital role in promoting normal brain activity and its disruption can ultimately lead to dementia. 

Chicken, meet egg. Egg, meet chicken.

So, from bacteria to human, carboxyl methylation is necessary for memory. Did these pathways evolve individually in parallel, or did the memory we have today originate in the predominant lifeforms found within us?

 

Suggested Reading

Li and Stock. (2009) Biol. Chem. 390: 1067-1096. DOI 10.1515/BC.2009.133

STORYTELLING IN SCIENCE: THE CELL AS YOUR FAVORITE RESTAURANT PART III

<img alt="Storytelling in science visualized through bacteria"      src="open-for-business.png">
Storytelling in Science visualized

Perhaps a running list of metaphors so far:

Restaurant: bacterial cell

Building: cell membrane

Doors: channels and transporters

Patrons: metabolites/compounds/substrates and products

Employees: proteins/enzymes

Managers: two-component proteins to regulate gene transcription

Employee list: genome

Copy machine: DNA replication machinery

So, in the last part our restaurant was going great and we opened up a new restaurant with the same employee list among other things. The two restaurants are now independent of each other and are free to act accordingly.

What if things change and times are not going as well? The overall number of patrons drastically decreases, not enough electricity (ATP) to run the restaurant or running water (redox potential)? What if disaster is about to strike? How can the restaurant prepare all the managers, employees, the building, the doors, the patrons for it?

Luckily the restaurant has a monitoring system that can quickly make sure the restaurant will be ready for anything that comes its way. The monitoring system can take snapshots of all data generated by the restaurant: power supply, water supply, patron count, employee count, conditions outside the restaurant like weather or competing restaurants. The monitoring system is the bacterial second messenger systems. With the support of the managers, the monitoring system can instantaneously keep track of all variables and make changes as needed.

The system is detecting the start of a drought. This drought will lower the number of patrons coming and going from the restaurant. The drought will also change the available electricity and water supply of the restaurant. The monitoring system sounds the alarm, a message is sent over the intercom for all the managers and employees to hear and react to. The intercom message alerts some managers to call in additional employees while telling others to stop their work. Some employees take on a new job in preparation for the drought. The intercom message is the bacterial second messenger cyclic-di-GMP. The entire restaurant begins preparations for the drought so it can survive until better times are present. Other than changes to managers and employees, some new employees are called in to prepare the building itself. Perhaps to change the number of doors. The employees may also change the exterior of the building to better withstand the drought like changing a wood exterior to a brick or stucco one. The brick or stucco are the exopolysaccharides, complex sugars on the exterior of the cell that can serve as protection or to help cells adhere to each other to ride out the hard times together. 

When times change, the restaurant has to be able to change with them. That is why these restaurants have been in business for ~3 billion years and still going strong.

Storytelling in science: Metabolic pathways as circus rings

My family and I recently went to a circus. It had one ring, and that was manageable. We have also been to a traditional three ring circus in the past. Personally, I felt there was too much going on at one time to enjoy all three rings at once. Each ring had skillfully trained performers doing their job for the enjoyment of the audience simultaneously. That is how a circus functions. Now imagine if you were able to observe a circus with more than 1000 rings. Imagine the complexity and the majestic choreography unfolding before your eyes. This is essentially what bacteria have been doing f0r millions of years with ease Instead of rings, these little circuses have pathways, a group of proteins/enzymes that all function together to perform a task. Like a circus, these pathways are not in isolation but instead many are performing at the same time. Even the “simplest” bacteria have over 500 pathways. Imagine trying to watch a 500 ring circus and understanding what is going on or being in charge of all 500 rings as they perform. Just because we don’t understand microbes does not make them simple, it makes us naive.

When sequencing a bacterial genome, computers and researchers try to connect all the dots. That is, they try to predict the role each gene/protein plays within that circus. For a bacterial circus with 5000 members (genes), only about one third of those can be assigned to a particular ring (pathway). This means a majority of members from a genome have a role we haven’t observe enough to classify its context. Now, imagine two thirds of KNOWN genes in KNOWN bacteria and the fact we approximately know 1% (or less) of the total number of bacterial species on, or in or above, earth. It doesn’t take long to discover that there is much more to discover in microbiology.

We as humans are beginning to utilize bacteria, or their pathways, to advance our civilization. Whether it is to clean up our polluted, toxic land or to advance medicine through fecal transplants, bacteria will play a much bigger role in the near future. Not bad for such small species. 500 rings or 2000 rings, these circuses are truly the greatest shows on earth!

bacteria, metabolism, pathways, microbiology

A 1500 ring circus from a typical bacterium.

Continuing on the theme that bacteria are Nature’s smallest circus, I want to highlight the most glaring problem with our knowledge of these 2000 ring circuses. We have discussed how proteins encoded by genes within a microbe’s genome often work together to carry out their function, i.e. pathways (or rings). To date, according to the NCBI genome site 4019 bacterial genomes have been sequenced to the point that we know the number of genes and proteins each organism contains. Moreover, this equates to 7,309,205 genes total or roughly 1818 genes per genome. These are astonishing numbers. To show our futility as experts of all things natural, over 30% of these genes are considered hypothetical or uncharacterized. In some genomes, these genes make up 60% of the total genes. These terms are a technical way of saying “hell if we know what they do”. Computers have recognized them as genes or open reading frames, however, the gene itself isn’t similar enough to known or characterized genes for scientists or computers to call it “the same”. If these gene products (proteins) functions are unknown, they cannot be assigned to a ring in the circus therefore making the largest ring by far in any bacterial circus the “unknown” ring.

Storytelling in Science: The Cell as Your Favorite Restaurant Part II

Recap: The restaurant is the bacterial cell, the employees are the proteins/enzymes that serve the patrons which are the compounds/metabolites.

Who are the bosses that determine which, and how many, employees are needed for each type of patron?

The restaurant managers have a very important job to perform. They have to make sure the right number of employees are available to help their respective patron. If the balance between employees and patrons is not well maintained, it could cause disaster for the restaurant itself. In a past post, I tried to describe how bacteria made decisions. One of the predominant ways was the use of two-component systems. For this story, think of the restaurant managers as actually two people who need to work well together. One identifies its respective patrons and the other makes changes to the number of employees for those patrons. It is this balancing act that helps the entire restaurant to work smoothly.

A successful restaurant will open up new locations. The same can be said for bacteria. If conditions are right, the cell will divide into two cells. As with a cell, restaurants have to make sure certain activities are undertaken to ensure the new restaurant will be exactly like the successful one it is copying. The success of this restaurant is based upon the ability to keep the employees happy (by having patrons to serve and not sitting around bored) and keeping the patrons coming in. To duplicate this success, the new restaurant should have a building exactly like the current one so the patrons will easily continue to enter and leave. The new restaurant will also need the exact employee list for the managers to call upon when needed. The employee list is the genome of the cell that encodes the proteins needed for survival. That would make the copy machine that duplicates the employee list the DNA replication machinery. This special restaurant building is state of the art. It can expand until it is roughly double its original size then place a dividing wall down the middle of the large building until the building becomes actually two buildings. Now the restaurant can serve twice the number of patrons with the same efficiency as before. Each new building has the same employee list and rough the same number of employees to start off with. Then the managers start their work identifying the patrons in the restaurant to make sure the employees are there to serve them.

The two buildings shake hands and go their merry way…ready to serve.

In Part III, I will talk about the intercom system that allows major changes to happen to the kind of employees needed for economic downturns.

Storytelling in Science: The Cell as Your Favorite Restaurant Part I

Many say storytelling in science is a great way to describe complex material in an understandable way for the masses. In this post, I will try to use an analogy to illustrate the complexity of a typical motile bacterial cell.

Microbial Physiology through Storytelling

If there is anything Americans know, it’s food. We are a nation obsessed with food and frequent restaurants on a regular basis.

Imagine your favorite restaurant as one huge bacterial cell.

When I travel to another city, I can’t rely on habit to guide me to a restaurant for dinner. I have to search for it while driving down the road. In order to know when I have found the restaurant I am searching for, I must rely on signs telling everyone what the restaurant is. The sign is a way to recognize and identify the building as i) a restaurant and ii) the specific type of restaurant. Bacteria do the same. They have ‘signs’ (proteins and other molecules) attached to the outside of the cell that lets other cells around identify what the cell is. I go into the restaurant through a door that allows patrons to move in and out of the building like bacteria have gates or channels that allow molecules to move in and out of the cell. Almost always, patrons are different leaving than they were when entering the restaurant; filled with yummy food they consumed and perhaps stopping to make a deposit in the waste room before leaving. Many molecules that leave a cell are different than those that enter. The workers of the restaurant have to keep track of the number of patrons entering and leaving the building to efficiently serve the patrons. Each employee has a specific job to do for very specific patrons. The employees have to identify their patrons and serve them as described by the bosses. Bacteria have an array of workers (proteins and protein complexes) that have very specific job descriptions depending on the patrons (substrates and product molecules) present in the cell. The restaurant survives by serving as many patrons as possible efficiently and correctly just as a cell must survive by responding correctly and quickly to everything in its environment.

Imaging Cancer Tissue One Chemical At A Time

Since listening to a Gordon Research Conference talk several years ago, I have been simply amazed by the applications developing with use of the mass spectrometer. Imagine being able to ‘see’ a specimen or tissue without use of any lenses or cameras. Not only ‘see’ it but also know the chemical makeup of each point at high resolution.

A new study published online for the journal Proceedings of the National Academy of Sciences explains the application of mass spectrometry imaging (MSI) to cancer tissue. Researchers at the Imperial College London have described a process to make this more applicable in a clinical setting without waiting weeks for a histological assessment by a specialist. This new technique will move cancer histology from the analog to digital age by identifying the actual chemicals within a sample instead of relying on structure.

Reference

Chemo-informatic strategy for imaging mass spectrometry-based hyperspectral profiling of lipid signatures in colorectal cancer. Veselkob et al. doi: 10.1073/pnas.1310524111