#xBio: Sticking the prenyl to pathogen proteins

(by biochembelle) May 13 2014

By many estimates, the end of the antibiotic age is upon us. Bacteria can divide so quickly and the prevalence of antibiotics is so great that they’ve evolved resistance to the drugs targeting their key pathways, like cell wall synthesis. But what if there was a new way to target certain pathogenic bacteria – not with broad-spectrum antibiotics but by targeting proteins in your own cells? That very well might be the distant promise of new work in protein prenylation.

Microbes are incredibly clever and have evolved many mechanisms for evading, reprogramming, and exploiting your body’s cellular responses. Certain types of immune cells, like macrophages, recognize bacteria as foreign and eat them, engulfing the microbes in specialized compartments, which are acidic and filled with proteases that destroy the invaders (this process is called phagocytosis). As they are being enveloped, some pathogenic bacteria inject proteins into the host cell, which will reprogram the host cell’s responses.

For this to work, though, some of the bacterial proteins might need to be modified by the host cell’s machinery to work. Such is the case with Legionella pneumophila, the causative agent of Legionnaire’s disease. When inhaled, these bacteria invade and multiply in macrophages found in your lungs (1 in figure). As they’re phagocytosed, the bacteria secrete hundreds of proteins into the macrophages, many of which localize to membrane compartments in host cells, and this process is quite important for proliferation of these bacteria (2). Basically, the bacteria secrete soluble proteins, which are tagged with a particular lipid group by host proteins called prenyltransferases (3). In eukaryotes, prenyl groups for proteins come in two flavors – farnesyl, a 15-carbon chain, or geranylgeranyl, a 20-carbon train. These prenyl groups can control cellular localization of the modified proteins, trafficking between cellular compartments, further post-translational modifications, and interactions with other proteins (4).

From Curr Opin Chem Biol - doi: 10.1016/j.cbpa.2012.10.015

The modified bacterial proteins contain a recognition code call the CaaX sequence, which is required for prenylation. Two host proteins catalyze most of the farnesyl and geranylgeranyl transfers in eukaryotes, and these proteins have been creatively named farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTaseI), respectively. The transferases add their prenyl substrate to a cysteine on the target protein (the C of CaaX). “X” represents the target protein’s C-terminal residue, which confers some discrimination between FTase and GGTaseI, although there is some overlap in recognition. The “a” residues between the target’s cysteine and C-terminal residues are quite variable. These are the same recognition requirement for prenylation of host proteins. In other words, there’s nothing particularly special about the bacterial proteins; it’s just another way that pathogens co-opt the host system.

So this process is important for one particular type of bacteria. But how widespread is host-dependent prenylation of pathogenic proteins? Elia Wright, a doctoral student in Carol Fierke’s lab at University of Michigan, set out to understand this. Wright started looking for pathogenic proteins that had a CaaX motif. She focused her search on proteins that bacteria secrete as they’re phagocytosed, because she wouldn’t expect many other bacterial proteins to encounter the host prenyltransferases. Wright’s first step was to determine whether the sequences she identified could be modified by mammalian prenyltransferases.

Generating full-length proteins for biochemical studies can be a laborious process, requiring lots of time and resources to generate a few milligrams of protein, sometimes after months of optimization. Short peptide synthesis, by comparison, is consistent and straightforward. Plus a robust biochemical assay using fluorescently labeled peptides allowed Wright to define the kinetics of peptide prenylation. She used peptides with the sequence of the last 6 residues of the putative targets (which includes the CaaX sequence) and a dansyl group attached to the N-terminus. She mixed a peptide with a purified prenyltransferase and its lipid substrate and then monitored the fluorescence of the peptide. Dansyl is sensitive to the hydrophobicity of its immediate environment, so addition of the prenyl group to the peptide increases its fluorescence. Using this approach, Wright identified many peptides derived from pathogenic bacteria that were prenylated in vitro. The catalytic efficiencies for the bacterial peptides and those of host substrates were comparable, suggesting that the pathogenic ones might be able to compete with host proteins.

This assay told Wright that human FTase and GGTase I could modify the peptides, but she wanted to know whether the results would hold in a cellular context. So she expressed short peptides (in this case the last 15 residues of candidate proteins) attached to green fluorescent protein (GFP) in HEK293 cells. GFP can be detected by fluorescence microscopy, and its localization is generally control by what’s attached to it. So it provided an easy way to track peptide localization in cells. If a peptide wasn’t prenylated, as was the case for the known negative control myc, GFP was distributed throughout the cell. The C-terminal peptide of H-ras, a host protein that's farnesylated, served as the positive control, localized to plasma membranes. In this system, Wright’s peptide candidates could also cause GFP to localize to cellular membranes and compartments consistent with prenylation – Golgi, endoplasmic reticulum, and, in one case, even nuclear membranes.

Wright’s results suggest that a wide range of secreted proteins from pathogenic bacteria can be modified by human prenyltransferases, and they can even compete with endogenous host proteins. Wright used peptide substrates, but sequence and structural elements beyond the CaaX sequence can alter prenylation. Expression of full-length proteins might provide additional insight, but most systems for manipulating mammalian cells result in overexpression that doesn’t reflect what happens in nature. Ideally researchers would like to use a proteomics approach, a method that uses mass spectrometry to provide sensitive, global analysis of both host and pathogen protein prenylation. First, they need a way to capture and enrich prenylated proteins. A group at Max Planck Institute demonstrated the proof-of-concept for this approach for a specialized prenyltransferase, Rab geranylgeranyltransferase.

The Fierke lab teamed up with the lab of Richard Gibbs at Purdue University to identify novel substrates that might work with FTase and GGTaseI. It’s proving to be a challenge, as these enzymes seem to be refractory to most substrate modifications. Wright notes, “We are making strides in understanding what part of the natural farnesyl and geranylgeranyl diphosphate [substrate] compounds are recognized and which areas of the molecule can be chemically altered without changing the specificity of the enzyme, which is something that most people disregard when using [e.g. tagged or taggable analogs].” Furthermore, Wright notes that they are testing their analogs against arrays of peptides, rather than one or two selected sequences, so they can understand how the analogs compare to natural substrates. Sadly Dr. Gibbs recently passed, but the Fierke lab continues the work they started together.

There’s a long way to go before we’ll really understand the broader relevance of pathogenic protein modification by host prenyltransferases. Wright started with L. pneumophila but is now expanding her studies to peptides from other pathogens such as organisms causing food poisoning and tuberculosis.  With a lot of patience, hard work, and a little luck, one day this might provide a new strategy for tackling tough infections.

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'Social' Scientists

(by biochembelle) Feb 05 2014

First off... holy wow. It's been nearly a year since I posted here. It probably goes without saying, that was not the plan. The past year has brought many changes and the next holds the promise of some more. Hopefully finding more time to post in these circles will be among them. Now. Onto business! I'm planning an event for my local postdoc association where I'll be introducing postdocs to social media use for scientists. I'm a big proponent of using online platforms for collaboration, outreach, and networking. But I'm curious: Where are scientists connecting online? I hope you'll take a few moments to fill out a quick survey. View Survey Comments are open for pontification on the subject. I would especially love to hear about interesting or clever ways scientists are using different digital resources in their research and their careers!

7 responses so far

Open Thread: Teaching & Learning to Write in Science

(by biochembelle) Feb 20 2013

In science, our careers are judged by the written word - dissertations, papers, grants, reports...

Of course, you can't write a good paper without good data. Although you can perhaps present good data in the absence of good writing, but bad writing can detract and even confuse the data.

Yet, in my experience, writing is often not a formal training element in postbac science programs. In my program, formalized training consisted of one course, which met one hour a week for one semester. As I recall, we covered the basic structure of scientific papers, went over a couple of examples, wrote an abstract, edited classmates' abstracts, and did a round of revisions. As I mentioned previously, most of the learning process was on the ground training - "The time is upon us. Go forth and write."

The iterative write-edit-revise approach worked well enough for me. In part, it worked because my adviser and I communicated reasonably well. I picked up on and integrated preferences through rounds of revisions. In part, it worked because I was pretty comfortable writing. I performed well on writing assignments in high school and college, and writing was something I did in my spare time. The writing style was different, but I understood the basic mechanics and structure. It wasn't easy, per se, but was certainly doable.

Sometimes, though, writing is a struggle - from the perspectives of writer, reader, and mentor. What then? Likely no single approach works for every person, every time. It's good to have options.

What resources do you turn (writers) to? For example, some grad programs and career development offices run intensive writing workshops, self-paced courses, and peer editing.

As a writer, what do you do to improve your work? Do you rely on revisions from mentors or colleagues? Do you look for outside help? Do you hire a professional editor? I typically rely on edits from multiple people, and I prefer if one is representative of the audience to which I'm writing.

As an editor (in the broad sense, e.g. reviewing a trainee's or colleague's paper), how do you change someone else's writing? Do you make sweeping changes? Do you provide comments and leave it to the writer to change? Personally I find the former makes it a little to easy to transform someone's writing style to your own. With the exception of typographical or grammatical errors, I try to stick with comments and, if I keep repeating particular points, provide global feedback on the paper.

What are your tactics?

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Remodeling Communication

(by biochembelle) Feb 02 2013

For a career reliant on clear communication, I'm sometimes struck by how poorly scientists (myself included) communicate. Often we hear this criticism when talking about scientists interacting with the public, but it also applies to our interactions with other scientists via manuscripts, grants, presentations, and even conversations. The why of effective communication is self-evident. The challenge resides in the how. Although my focus here is writing, the ideas can be extended to other forms of communication.

Making the mold

How do scientists learn to write?

Looking back on writing in grad school, I realize that it was a "learn by example" and "learn by doing" experience. I had read many papers in the field and published from my PhD lab. I knew the basic structure of a scientific manuscript. My adviser and I would agree on an outline or framework built around figures. And then it was time to go forth and do. I would write. Someone would critique and edit, and I revise. Rinse. Repeat. Submit after n iterations.

The method can work well - or not.

For better or worse, our templates (i.e. the papers in our lab and field) impact our writing. Even when refraining from copy-paste, you could likely identify which lab published a paper based solely on the first lines of the abstract and introduction. On one hand, there are limited ways to introduce our system; on the other, we may simply follow the road taken by our predecessors without much consideration for alternatives.

Likewise, the editing process shapes our present and future approach to communication. We each have personal style preferences - particular words, phrases, and sentence and paragraph structures that we love or hate. We don't edit solely for clarity and effectiveness, but rather we tend to edit in the context of our preferences. The categories overlap but are not mutually inclusive. I think using word processing tools can exaggerate the influence of individual preferences. It's easier to rewrite sentences and even entire sections,as  compared to cramming extensive edits in the margins or between the lines of a printed page. Whereas using pen and paper we might scribble a note about clarity or verbosity, with a word processor, we might succumb to the temptation to simply "fix" the paragraph ourselves.

In the end, we might not necessarily learn the general mechanics of communication. Rather we learn to emulate a style of writing specific to our advisers and peers.

Breaking the mold

We create a mold and use it. Papers are accepted, and grants (hopefully, eventually) funded. The mold works. Why bother changing it?

Why not? Sometimes our communication needs and goals change - pursuing a different branch of a project, establishing new collaborations, connecting with the public, teaching new students - but there's also intrinsic value in improving what works.

Yet changing our writing is hard. We've adopted certain patterns of communication, learned early in our careers and enforced by daily interactions. Some call it "structural priming" or "syntactic persistence", and we have to work to break away from it. How we break the mold depends, to an extent, on what we want to accomplish, but ultimately we must do things that challenge our approach to communication. Here are some things I'm trying.

Read broadly. It's a daunting prospect in context of publication volume, but if we want write differently, we need to read different things, instead of reading the same authors repeatedly. If we write for a new target audience as we would for the old one, we can miss them altogether. We have to consider what works for our new target, and a good way to learn is to read what they're reading. I think this is especially important when our work moves in new directions. Likewise, if we want to reach a non-expert audience, we should be reading effective science communicators to find out what works. (National Geographic's Phenomena 'salon' is a great place to start.)

Take note of style, not just data. As scientists, when we read a paper or listen to a seminar, we typically focus on data because we're evaluating the quality and applicability of new findings. Regarding style, I think we often consciously note things that make results very difficult to understand, but we rarely actively discuss what makes a presentation particularly engaging. A few months ago, I was reading a paper from Susan Lindquist's lab. There was a certain elegance in the writing that made the science clear and interesting. I mentioned something to this effect on Twitter, and someone asked what made a technical paper a joy to read. I had to think about it. Both obvious details and subtle abstractions come into play.  Several people offered their thoughts, which were collected by Doctor Zen.

Learn more about communicating. Scientists are experts but not about everything. Just as we have invested immense amounts of time in understanding our favorite system and discipline, many others have devoted their time to investigating, applying, and teaching effective communication. Although technical writing may differ in ways, we stand to learn from that expertise. Consider this discursive on zombie nouns or The Writer's Diet test to help identify padding in your writing. Denise Graveline, a communications consultant, shares lots of tips and links at don't get caught and The Eloquent Woman.

Try new things. Some exercises can force us to step outside our routine. We might spin a well-trodden missive into a grim fairytale. We can use platforms that impose limits. Twitter calls for brevity and, in lively threads, rapidity, but the language can be simple or technical. The Up-Goer Five Text Editor provides the inverse experience - use only the top 1000 most used words in English, but as many times as you like. (The sister Up-Goer Six Editor permits use of all words but colors them by how commonly they're used.) Many researchers accepted the challenge to explain their work with Ten Hundred Words of Science. The products of such exercises may not be adequate or appropriate for what we need to accomplish, but they call on us, at least fleetingly, to change how we write, to examine how we use language, and to understand where we can trim or modify language in discourse.

Breaking habits that haunt our keystrokes isn't easy. And despite my opening statement and our collective reputation of being bad communicators, I think many scientists are actually quite deft with different forms and different audiences. But to continue being good, we have to keep improving. How are you reshaping the mold?

2 responses so far

Lighting the Way

(by biochembelle) Nov 11 2012

"You are found guilty as charged. You shall be cast out with your family, into the darkness, to live out the rest of your short life. And miserable it shall be be without the Light." The oracle's voice echoed through the chamber.

The final reverberations stirred the man. "But-"

His protestations were cut off as the oracle boomed, "You have made your case, and in doing so, it is clear that you have abandoned the Light. It is only just that the Light now abandon you. Remove him from the court." The last was addressed to the guards, who immediately came forward and drug the man away. His unheeded protests were blocked out as the heavy doors to the chamber banged shut.

The oracle spoke more quietly. "There is only one case left to attend."

Those gathered in the chamber looked about curiously. There were no opening of a door to usher in another offender. This was very odd indeed.

The oracle remained silent, but he raised his arm and pointed at a woman in the crowd's midst. Those around her glanced to their left and right, building their confidence that they were not being singled out. Then the eyes turned to her. The oracle beckoned, and she slowly made her way forward until she was standing a few meters away.

The oracle gazed down at her haughtily. "You understand why you are here."

"Not entirely," she replied meekly.

He sniffed disdainfully. "You have been a keeper of the Light for years. You have worked diligently to understand the Light. And yet, we learn that you reject one of the central tenets of the Light, the truth of how the world and all that lives within were formed."

She shifted from one foot to the other. "Well... yes. It is how I was tau-"

He spoke over her. "You have confessed your crime. You are found guilty, betrayer."

She interjected. "But it doesn't change who I am or what I have done."

"Silence!" the oracle shouted. "How could any student of the Light believe such nonsense? It calls  you are and everything you have done into question. You are to be exiled into the darkness. It is the only suitable recourse." He motioned for another pair of guards to come forward. They escorted her from the chamber, but unlike her predecessor, she was dumbstruck and did not utter a sound.



The establishment and enforcement of "truth" can take on disturbing tones. We need only look back through history at the individuals ostracized by religious institutions and society for their scientific findings and theories. Even today, we witness highly polarized debates over big issues that should be driven by science - origins of life, climate change, genetic engineering, vaccines...

Scientists have an obligation to get involved in these debates, to inform the public, to see that solid science drives policy. Scientists are sometimes stereotyped as emotionless automatons, but these debates can stir the passions of scientists as much as they do the opposition. We can be so consumed by the rightness of our cause that we respond with the certitude and hubris that we condemn in others.

Some may say, "Well, we have facts and data on our side. We have the right to speak with authority and the responsibility to educate the masses." I say, though, that how we do so matters, and if we're not careful, we risk alienating those we are trying to reach.

It is remarkably simple to state, "Creationism is ludicrous. Science proves it." or "How can you possibly ignore human impact on climate change? The data are clear!" It's easy to respond to "anti-science" sentiments with snark or an aloof dismissal.  admitting a contrary belief or even ignorance on an issue is oft met with derision rather than a desire to share knowledge. I have observed and experienced the attitude firsthand, and it does little (and I would go so far as to say, nothing) to advance the discourse.

Even backing a position with evidence may not have the intended impact. Coming out of the gate laden with disdain for a group's or an individual's stance creates barriers to true communication. "You're wrong, and here are the reasons you are wrong." This is not a position that invites engagement. It can easily move beyond establishing authority and into projecting superiority. Interest in active listening and participation wanes when someone perceives a feeling of inequality. It's no longer about discussing differences; it's about winning - for both parties involved.

We liberally apply the label "anti-science" to creationists, anti-vaxxers, and so on. But I suspect that most people who hold beliefs contrary to evidence are not actually "anti-science". They often have a deep interest, and even at times an education, in science. But the issues push back against deep-rooted beliefs, and it takes much more than saying, "Well, this is how it is." to change those. In some ways, we've been asking people to trade in one set of fundamental doctrines for another because Science says so. Yes, there are experiments and data to affirm scientific theories and conclusions, but how well are we explaining the process? And do we take for granted the base level of scientific literacy students coming out of high school and college? Perhaps these are among the reasons I adhered to creationism for so long (even well into a PhD in the sciences) and why I still don't have a good understanding of evolution to this day.

The transfer of knowledge takes time. On some issues, we are working against years of ingrained instruction, the rejection of which carries weightier consequences than failing a test, for some. On others, we have to push back fear, propaganda, or distrust of industry in science. These are not barriers that can be overcome in minutes, or an hour, or even a day. It could take months, even years, to make progress. Patience brings no guarantee of success, but it does offer a chance. If we really want to change the world's - or even one person's - understanding of science, we need to be ready for a long haul. And that means getting them back to the table, the bar, or wherever we might meet time and again.


Related & Recommended - Should Scientists Promote Results over Process?

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Campaign season! For organelles?

(by biochembelle) Oct 31 2012

Outreach is a strong theme among the online scientific community. We talk about the importance of engaging the public, talking to politicians, and interacting with students. In essence, outreach is about making connections with people outside our field and, in some way, sharing the wonders and importance of science.

Outreach can present many challenges for scientists, one of which is finding opportunities to engage in outreach. We feel we lack time, ideas, support, audience... But with the rise of social media use, new opportunities open up, and sometimes without any intent, we can stumble right into them.

I am very active on Twitter. It's a way for me to stay connected to the science online community, even when I'm short on time or energy to keep up with the avalanche of blog posts and comments the sphere puts out every day. I follow many scientists all over the map, with regard to geography and disciplines. One of them is Anne Osterrieder, a Research and Science Communication Fellow in Plant Cell Biology at Oxford Brookes University, and last week, she started reposting some rather curious tweets. They were from accounts named after cellular organelles, which seemed to be touting their supremacy among organelles or attacking others for the problems they create.

Anne discovered that these tweets were coming from an AP biology class in Illinois.

Two of my favorite organelles, lit with fluorescent labels

The core idea originated with Marna Chamberlain, a teacher at Piedmont High School in California: Get students to run campaigns for a cell organelle - posters, speeches, flyers on why theirs was the supreme organelle.

Brad Graba at William Fremd High School in Illinois decided to run with it and extended the campaign to include Twitter.

Then something happened that Brad Graba did not intend, I think. Alerted by Anne (who posted a great early intro), scientists began joining in under the hashtag #organellewars. (If you're unfamiliar with Twitter, a hashtag is simply a handle for tagging and finding related messages.) Before long, organelles were discussing their functions and failures with scientists around the world. The project was even mentioned on BBC Radio!

It's clear from the Twitter campaign that Mr. Graba has some smart and creative students to teach. But I think it also shows that cool and unexpected things can happen when you begin to integrate social media with education and outreach. Thanks to Mr. Graba and his class for letting us crash their project! I hope they gained something from the experience. I know I certainly did! 🙂

Oh, and one more thing: It's time to get out the vote! You can vote for your favorite organelles during the Mr. Graba's AP Bio class periods today (one is up now [9:45 am, EDT] and others will be up at about 10:15 and 2:50).

2 responses so far

One moment

(by biochembelle) Oct 07 2012

You won't have much time.

All you have is a moment...

So little time to connect.

But that moment can change the game.

One brief moment might be all you get...

but one spark can kindle a flame.

Or you might capture them with that moment...

and keep them engaged until the job is done.

Then again maybe nothing changes.

An interaction with no response.

A random collision, and you both wander away.

 This is how biology happens.

Cells, proteins, DNA, and other molecules crashing into one another... Sometimes they stick, others they don't.But what happens in those moments can change the system - upset or restore balance, redirect outcomes.


Human interactions are not so different. Connections are made in moments.

Or not.


Whatever happens in that moment defines what happens in the next.

In biology, transient interactions are destined for specific outcomes. Factors may change them - promote their initiation, amplify their output, prolong their existence - but even the tweaks are hardwired.


What if you could influence that moment?

Human interactions are not so deterministic. For that, we may be grateful.

Or perhaps wistful.


What if you had the potential to shape what happens in the next?

From random encounters in a crowd to introductions by colleagues to the opening lines of a narrative, every connection begins with one moment.


What if you could extend your influence?

We have opportunities to connect with people at every level, to share the import and intrigue of science (or anything else).


All you have is a moment.

But if we want them to be engaged, to stay for the end of the story, to return for more, that first moment can make all the difference.


What happens in yours?

7 responses so far