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Lorne Proteins 2020

Sat, 08 Feb 2020 05:19:01 +0000

Lorne Proteins 2020

If you’ve landed here after checking out my poster , welcome. If you stumbled here by accident, the more the merrier. Either way, thanks for stopping by!

I hope you found something interesting or useful, and enjoyed hearing about the work we are doing to develop new tools for measuring proteostasis. If you have any questions, want to know more about what we do, or simply want to get in touch, you can find me on twitter head over to the .

For anyone who wasn’t at the conference, or didn’t get a chance to say hi, I have included a brief run-down (and tried to skip over most of the gory – boring, technical – details!).

What’s all this proteostasis business?

Cells have an extensive quality control network responsible for maintaining their molecular machines, including synthesis, folding, degradation and transport

\[1\]

. Collectively, this machinery is known as the proteostasis network. Proteostasis imbalance results in protein misfolding and aggregation, the central molecular signature of neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

We lack knowledge of which proteins in the cell become vulnerable to improper folding during proteostasis imbalance. One measure of protein foldedness is the extent to which a protein can be unfolded thermally or with a chemical denaturant such as urea. Until recently, it has been difficult to track foldedness in cells due to sheer complexity of the many ten’s of thousands of proteins and proteoforms needed for our cells to function.

To overcome this, this work specifically aimed to:

Devise a chemical biology and proteomics approach to monitor the foldedness of the proteome.
Determine the influence of pharmacological agents that unbalance proteostasis on proteome foldedness.

What is tetraphenylethene maleimide, other than a tongue twister?!

Tetraphenylethene maleimide (TPE-MI) is a dye (tetraphenylethene maleimide) that becomes fluorescent when it binds to reactive thiol residues

\[2\]

. Buried, reactive thiols are the most buried residue of all amino acids in globular (properly folded) proteins. Monitoring the exposure of these thiols using TPE-MI gives us a sensitive way to probe foldedness in complex mixtures.

To test this strategy, we used a purified, well-studied protein – β-lactoglobulin. We know both the sequence and 3D structure of this protein, and its behaviour in a range of denaturants is known. β-lactoglobulin has 5 thiol residues, of which two pairs are bonded and the remaining one (Cys121) is buried in the core of the folded protein. As β-lactoglobulin unfolds in increasing concentrations of denaturant, we get a corresponding increase in TPE-MI fluorescence.

But what about the giant protein soup our cells make, I hear you ask…

While TPE-MI can reliably tell us about the unfolding of a single, purified protein, our cells are jam-packed with thousands of copies of thousands of different proteins. How can we possibly know which proteins TPE-MI is binding to?

Enter: proteomics. This revolutionary tool is a workflow based on mass spectrometry which can report back on the composition of complex mixtures – both which proteins are present, and the relative amount of that protein. The basics of this technique are beyond the scope of this post, but if you are looking for more details check out the resources section below

\[3-5\]

. I have included a brief explanation of the method we use below, which relies on isotopically labelling cells in culture before lysing, denaturing and labelling with TPE-MI. Proteins are digested (chopped up) into regular, smaller pieces before analysis via mass spectrometry.

Using custom software, we can identify and quantify protein pieces. Unfortunately, we cannot identify the protein pieces specifically labelled with TPE-MI, but instead, we look for the loss of those pieces. We can do this for many many proteins and start to understand the concentration at which specific proteins unfold. We can even start to understand intricacies in unfolding for separate protein domains.

How does this help us understand disease mechanisms?

This new method now provides a way to monitor how the stability of proteins (measured how much denaturant it takes to unfold them) changes under conditions of proteostasis impairment that are present in disease. For example, we are now investigating the changes in proteome foldedness when we inhibit hubs of the proteostasis network such as molecular chaperones. How do the proteins that rely on specific molecular chaperones to be folded change when those proteins are no longer active? What if we stop the cell from degrading old proteins? Or from decorating their proteins with extra modifications that mediate structure and activity like phosphorylation? Defects in this machinery are common in neurodegenerative protein aggregation diseases and we now have the tools to start tackling these questions.

References and handy links

Chiti F, Dobson CM (2017) . Annu Rev Biochem 86: 27–68.
Chen MZ, Moily NS, Bridgford JL, Wood RJ, Radwan M, Smith TA, Song Z, Tang BZ, Tilley L, Xu X, Reid GE, Pouladi MA, Hong Y, Hatters DM (2017) . Nat Commun 8: 1–10.
Lottspeich, F. (2009). . In Proteomics (pp. 3-10). Humana Press.
Graves, P. R., & Haystead, T. A. (2002). . Microbiology and molecular biology reviews, 66(1), 39-63.

That’s all from me for now. Still can’t get enough or want to know more? Simply want to get in touch? Find me on twitter head over to the - love to hear from you! Now, back to the beach 🏄🌴☀️ !

Toolbox Talk: Scientific posters

Tue, 30 Jul 2019 10:39:57 +0000

Going to a large, often international, conference jam-packed with superstars in your field is one of the privileges of being a scientist. Conferences are essential for building your network, keeping up-to-date with the progress inside your niche and more broadly in your field, and establishing or maintaining collaborations. If you’d like to know more about my conference experiences, check out . I was lucky enough to attend and present short talks at a few conferences in 2017, and it was a wonderful opportunity to show the scientific community what I have been working on.

However, there are only so many oral presenter slots available at such events, and often these are used by the organising committee to highlight the very best and newest science. But what about the rest of us? It can be really difficult to get the most out of a conference, especially as a student, if you don’t have an opportunity to showcase your work. Enter: poster presentations.

Wait, like in elementary school?

If you are thinking of a giant piece of butcher’s paper, covered with scribbles in multi-coloured permanent markers, you aren’t too far wrong. But things have evolved a little since the school days, and we do have slightly more developed tools for displaying our work these days. Unfortunately, making an engaging scientific poster is often not something we are formally prepared for during our scientific training. We are being moulded into the next generation of scientists, after all - what no one tells you is that artist is just one of the many additional hats that you will wear often as your career progresses.

With this in mind, I thought I would share a little about my process and tools for making an engaging poster. It is by no means a one-stop-shop, as every time I make a new poster I learn a little more about the process, but hopefully, you will find something useful as you prepare for that next science-filled conference adventure.

Tools

There are plenty of purpose-built design software packages out there specifically for making commercial posters, and some even targetted at producing scientific visuals. I have included a few links in the additional resources, but to be honest, I have had mixed success with these - if you find any that I have missed or that you’ve found particularly helpful to let me know!

In the meantime, I have settled on a combination of a freely-available vector graphics program (Inkscape) for compiling individual panels into standalone figures and presentation software (e.g. or ) for the overall poster design. If you haven’t heard of vector graphics programs before, or are unsure why you might want to use one, you can read more . In essence, because of the way they are constructed, vector graphics are easy to manipulate and can be scaled indefinitely without losing their quality as they are not composed of pixels, in contrast to the raster-based file types jpeg and png.

For data-laden figure panels, I will usually stick with python or (if you are interested in this, check out some of my other toolbox posts ). For any work dealing with protein structure, is your friend. The learning curve can be quite steep (I am only just now mastering the basics of opening a structure file, editing the colours and changing the display type from ball and stick to ribbon!) but it is relatively easy to produce a striking visual that will aid in any handwavy descriptions of a protein’s structure. Keep an eye out for my beginner’s basic guide to PyMol in a future Toolbox Talk!

Last, but definitely not least, I use a to create a link to this website. Even if the name is not familiar, I am sure that you would have come across in the wild before - they are a type of matrix barcode that links to a website or application when scanned with a smartphone camera. There are plenty of free online platforms for creating your own QR codes, and many that give you the ability to adjust the shape and colour of the individual elements, however be careful to know the difference between static (meaning you have no flexibility to adjust where the link directs after printing) or dynamic (that contain an automatic redirect which you can adjust after printing) to make sure you get one that suits your particular needs. I have found this especially handy for posters - by encoding your contact details or link to your website, it is easy for conference attendees to get in touch without having to hastily scribble down your email on their crumbled napkin.

Techniques and tips

Even the most exciting and life-changing data can find itself on a poor poster. There are many unwritten (and written) rules for making an engaging and eye-catching scientific poster, and many more experienced people than I have written guidelines and tips, some of which you can find in the resources section below. In any case, I thought I would throw in a few of my trusty tips here:

Layouts should be simple and easy to follow. I find that creating boxes to guide the reader through the poster in a logical fashion can be helpful. Remember a poster is about walking someone through your science and stories that jump backwards and forwards can make it hard to stay engaged. As a general rule, try not to require your audience to do too many optical gymnastics to follow the narrative of your science. When creating a new poster from scratch, I like to sketch out the layout, brief text tidbits and figure ideas by hand in A4 format to help get a handle on what data I need to track down and figures I need to produce. I also find drafting this initial layout in A4 size helps to keep my final poster from getting too overcrowded.
Find SVG versions of logos for the institutes that contributed to the work. It is good practice to acknowledge the financial support any project receives, and posters are no exception. In practice, I typically will have anywhere from 1 - 3 logos on my poster and any additional smaller contributions can be acknowledged in the text if you feel it is necessary. I will always find SVG versions of the logos, such that they can be edited and resized without losing any quality. Generally, you will be able to access these from your University brand team, or a quick google search with an advanced filter to find SVGs works well too.

Never underestimate the power of a good colour scheme. There is nothing worse than a poster that looks more like a rainbow kaleidoscope than a scientific document. Try to steer clear of elaborate colour schemes, and away from anything too light or bright. Also, keep in mind that picking a simple colour scheme to use throughout the entire poster (e.g. one type of sample is the same colour in all panels) helps to provide continuity for your reader. For those who are colour-coordinator challenged, a quick google search for colour palettes will provide all the inspiration you need.

Remember the power of size, shape and colour for drawing attention. With the limited time and large amount of information available at poster sessions, conveying the most important points from your work is crucial. Using accent colours, bold fonts or larger sizes can help to draw your reader’s eye to the key aspects of your work, and hopefully prompt them to ask more about the details!
Consistency is key. A poster, aside from displaying scientific results and information, is at the end of the day a piece of art. As with any beautiful artwork, the little things matter! Use the handy aligning and specific dimension setting functions in powerpoint to make sure everything is balanced and consistent. Check that the text sizes, fonts, line widths etc are identical throughout your poster, especially for things like headers and panel outlines. There will always be one or two typos that sneak past you, but it’s also a good idea do several final read-throughs to check for typos - once your masterpiece is printed, there is no turning back!
Print smaller versions for proofing. Printing an A4 version of your poster is a great way to check for readability and those last-minute alignment or spelling errors. As a general rule, anything that is not readable in this format will not become more-so simply by being printed bigger - remember you want to be able to engage your visitors in conversation, not have them glueing their nose to your poster trying to make out the details!
PDF is your friend. Particularly if you include transparent artwork or special characters, it is good practice to export your poster from powerpoint to PDF before submitting for printing. There are also a few tricks with powerpoint to ensure the quality is maximal, which can be found in the ‘Options’ menu of the dialogue box during export.
Be prepared to print early. Some institutions have their own print services, and these can be a cost-effective way to have your poster printed. I have a lot of experience with Officeworks and have generally been thrilled with their service, although I would recommend visiting a store in person to submit and collect your poster. They will generally give you a test print which is handy to make sure your creation survived the file format change. Regardless of which route you choose, plan to have your poster finalised and printed several days before departure - this way you will have a few days up your sleeve to handle the inevitable curve-balls!

Useful resources

from Animate Your Science has some straightforward tips for layout and content. I will admit to disobeying a few of their “rules” but generally great advice!
Toolbox talk on and the

Did I miss your favourite tool or technique? Find me on or head over to the to tell me more!

Image credits: codytdavis, greyritualstudio, alecuffia and arterbury via

#LorneProteins2019 & #FASEB2019

Mon, 11 Feb 2019 06:07:09 +0000

If you’ve landed here after checking out my poster or the , welcome. Thanks for stopping by!

Just can’t get enough of the pretty pretty science? You can find details about the work I presented on my poster here. For anyone who wasn’t at these conferences, or didn’t get a chance to say hi, I have included a brief run-down (and tried to skip over most of the gory - boring, technical - details!).

Now - onto the science!

Setting the scene.

Cells have an extensive quality control network responsible for maintaining their molecular machines, including synthesis, folding, degradation and transport

\[1\]

To overcome this, this work specifically aimed to:

Devise a chemical biology and proteomics approach to monitor the foldedness of the proteome.
Determine the influence of pharmacological agents that unbalance proteostasis on proteome foldedness.

What is tetraphenylethene maelimide, other than a tongue twister?!

Tetraphenylethene maelimide (TPE-MI) is a dye (tetraphenylethene maelimide) that becomes fluorescent when it binds to reactive thiol residues

\[2\]

To test this strategy, we used a purified, well-studied protein - β-lactoglobulin. We know both the sequence and 3D structure of this protein, and its behaviour in a range of denaturants is known. β-lactoglobulin has 5 thiol residues, of which two pairs are bonded and the remaining one (Cys121) is buried in the core of the folded protein. As β-lactoglobulin unfolds in increasing concentrations of denaturant, we get a corresponding increase in TPE-MI fluorescence.

But what about the giant protein soup our cells make, I hear you ask…

Enter: . This revolutionary tool is a workflow based on mass spectrometry which can report back on the composition of complex mixtures - both which proteins are present, and the relative amount of that protein. The basics of this technique are beyond the scope of this post, but if you are looking for more details check out the resources section below

\[3-5\]

Using custom software, we can identify and quantify protein pieces. Unfortunately, we cannot identify the protein pieces specifically labelled with TPE-MI, but instead we look for the loss of those pieces. We can do this for many many proteins and start to understand the concentration at which specific proteins unfold. We can even start to understand intricacies in unfolding for seperate protein domains, such as in the example below where different reactive thiols give information on specific domains of Hsp70.

How does this help us understand disease mechanisms?

References and handy links

Chiti F, Dobson CM (2017) Annu Rev Biochem 86: 27–68.
Chen MZ, Moily NS, Bridgford JL, Wood RJ, Radwan M, Smith TA, Song Z, Tang BZ, Tilley L, Xu X, Reid GE, Pouladi MA, Hong Y, Hatters DM (2017) Nat Commun 8: 1–10.
Lottspeich, F. (2009). In Proteomics (pp. 3-10). Humana Press.
Graves, P. R., & Haystead, T. A. (2002). Microbiology and molecular biology reviews, 66(1), 39-63.

That’s all from me for now. Like the pretty pictures? Still can’t get enough of the science? Simply want to get in touch? Find me on twitter or head over the to page - I’d love to hear from you! Now, back to the beach 🏄🌴☀️ or the snow-covered mountains 🏔️⛷️!

Magnified 2018

Tue, 05 Jun 2018 07:53:54 +0000

The Illawarra Health and Medical Research Institute (IHMRI), in association with the School of Arts, English and Media at the University of Wollongong (UOW), are hosting a fundraising exhibition featuring images from the work of researchers and students, in support of Motor Neuron Disease (MND) research at IHMRI. This is a cause quite close to my heart, and as an alumnus, I was lucky enough to be invited to contribute. I actually had a heap of fun choosing my artwork (you can check out my submissions below) and for all the details on when and where you can find the exhibition head . Works in the exhibition will be on sale to the general public, with all proceeds going towards Motor Neuron Disease (MND) research at IHMRI. What better reason do you need to pick up some fantastic sciart?

Decoding Proteostasis

Proteins are the major cellular building blocks fundamental for most basic operations sustaining life. Most proteins need extensive management by the cell’s proteostasis machinery to maintain their proper shape. Proteostasis imbalance can cause neuronal death, leading to neurodegenerative disease. Various cellular stresses, such as inhibiting housekeeping functions or dismantling structural components, can differentially impact neuronal health. Understanding changes in each individual protein (columns) within a cell as it responds to different stresses (rows) highlights how different stress types influence proteostasis. Revealing common vulnerable pathways, by decoding this ‘proteome barcode’, will help focus future therapeutic targeting on neurons weakest links.

The twists and turns of proteostasis

Misfolded proteins aggregate into elongated fibrils (yellow/green ribbons), which cause neuronal death and lead to neurodegenerative diseases like MND, Parkinson’s and Alzheimer’s disease. Using powerful microscopes, we can watch how the molecular chaperones (red), part of the cells proteostasis defence network, attach along the fibrils, helping to protect the neurons from these toxic tentacles.