Posts Tagged ‘thesis’

My Thesis: Wordled

Dec 17th, 2010

When I turned my 2010 facebook status updates into a word cloud, I was unsurprised to see the word thesis looming large at the centre. It has certainly been one of the defining elements of the year for me, and facebook was a good outlet. By contrast, my twitter feed was probably more dominated by the election, and the associated politics; I can’t know for sure, as ‘2010 in statuses’ didn’t seem to look back beyond the past few days.

On seeing my facebook status cloud, a friend, who has had a similarly thesis dominated year, suggested turning my thesis into a word cloud. So I did.

Thesis Wordle

A word cloud of my thesis

I was amused by the huge ‘et al’ in the centre, a suggestion that perhaps I has spent more time talking about other people’s work than my own. The rest of the cloud is quite pleasing, an abstract in single word chunks. The dominance of the slightly generic ‘protein’ and ‘proteins’ is perhaps unsurprising, yet I am surprised at how comparatively small ‘gene’ and ‘genes’ are. It is a weighting which is entirely consistent with the focus of the thesis, but still feels at odds with my background in genetics.

Mitochondrial proteins at the proteasome, are you MAD?

Dec 16th, 2010

About this post

This post was originally written for a job application, however I was never able to use it. It covers a growing field closely related to the subject of my PhD. As the article was originally intended to be written for a blog of a biology journal it is primarily written for those with training in the biological sciences, although I also tried to make it accessible to an informed lay audience. Unfortunately I think this attempt at pitching the article at a wide audience hasn’t been entirely successful, at times seeming too simplistic fro the primary audience, and at others too inaccessible to a lay audience. I also feel the article could do with being less formal, however given the original purpose of the article I felt it was better to lean towards too formal, than risk being perceived as too informal.

I should clarify that I don’t discuss any of my work in this article.

When proteins become damaged or are no longer required by the cell, they can be broken down into their component peptides, allowing these resources to be reclaimed. Much of this recycling occurs at proteasomes, hollow barrel like structures located in the cytoplasm and nucleus of the cell. Breakdown of proteins occurs in a catalytic chamber at the centre of the structure, and entry of potential substrates is tightly regulated by caps at either end of the chamber.

In most cases, a protein’s fate at the centre of the proteasome is determined by the attachment of a chain of ubiquitin: a small protein, which can be covalently attached to lysine residues on other proteins. This chain is recognised by components of the proteasome, as well as by shuttle factors, which help recruit substrates to the proteasome.

Proteins within the cytoplasm or nucleus have easy access to the proteasome, however those within membrane bound organelles must first be translocated into the cytosol. The first evidence for such processes occurring was found in association with the endoplasmic reticulum.

Endoplasmic reticulum associated degradation, or ERAD, describes the process by which unwanted proteins within the endoplasmic reticulum are retro-translocated to the cytosol. Here they are tagged with ubiquitin, and delivered to the proteasome. ERAD relies on the co-operation of a large number of factors. A series of chaperones identify proteins which are undergoing difficulty folding and in turn they deliver them to complexes in the ER membrane. From here, proteins are transferred to the cytosolic face of the organelle, and chains of ubiquitin are attached, marking them for proteasome mediated degradation. The hydrolysis of ATP by the hexameric Cdc48/p97 provides the energy to extract ubiquitinated proteins from the membrane, and co-factor proteins ensure efficient delivery to the proteasome or its shuttle factors.

For a long time it was assumed that no such system existed in mitochondria, the respiratory centres of the cell, as they have their own complement of proteases. However, increasing evidence is suggesting that the proteasome may play an important role in the degradation of some mitochondrial proteins, leading to the proposition of mitochondria associated degradation, or MAD.

Traditionally, the degradation of mitochondrial proteins has been seen to be the domain of the mitochondrially localised ATP dependent proteases. The inner mitochondrial membrane contains multi-subunit proteases with their catalytic activity orientated to face either the inter-membrane space, or the mitochondrial matrix. Additional proteases, such as Lon/PIM and ClpXP, are found within the mitochondrial matrix. This collection of proteases is sufficient to provide protein degradation in the mitochondrial matrix, inner mitochondrial membrane and the inter-membrane space, however analysis of the products of proteolysis indicate that inter-membrane space proteins are under-represented.

Some of the earliest evidence of proteasome dependent degradation of mitochondrial proteins came in 1985, when Rapoport et al.1discovered that some mitochondrial proteins were ubiquitinated, and degraded in an ATP dependent manner. Subsequent to this, other ubiquitinated proteins have been found to localise to the mitochondria, and appear to undergo proteasome mediated degradation.

More direct evidence arose when Margineantu et al.2 found that the heat-shock protein Hsp90 appeared to promote ubiquitination of OSCP, a matrix localised subunit of the mitochondiral F1F0-ATPase. Further investigation revealed that disruption of Hsp90 resulted in reduced turnover of OSCP and other mitochondrial proteins, resulting in accumulation on the outer mitochondrial membrane; similar changes were observed on proteasome inhibition. This accumulated protein appeared to be a result of retro-translocation of mature mitochondrial proteins, indicating that mitochondria possessed a pathway of retro-translocation and proteasome mediated degradation, analogous to the ERAD pathway.

Dissection of the mitochondria associated degradation pathway is still in its early stages, and as yet many of the required components remain speculative. Comparison with the better characterised ERAD pathway is inevitable, and it remains to be seen if any elements will be shared between the two pathways. Recent work by Heo et al.3 discovered that Cdc48/p97 was recruited to mitochondria in a stress responsive manner by a protein they called Vms1 (VCP/Cdc48-associated mitochondrial stress-responsive). Disruption of this process resulted in a reduction in ubiquitin-dependent degradation of mitochondrial proteins, leading the authors to propose that Cdc48/p97 may perform a key role in MAD, as well as in ERAD.

The highly oxidative environment of the mitochondria means that its proteins are particularly prone to damage. Mitochondrial proteases have long been known to provide one line of protein quality control, and it is increasingly apparent that the proteasome dependent degradation of the MAD pathway may provide another. Dissection of the MAD pathway will be essential for forming a complete picture of the mitochondrial protection against damaged proteins, and may provide insights into diseases such as amyotrophic lateral sclerosis, which are associated with an accumulation of misfolded proteins in the mitochondria.

  1. Rapoport, S., Dubiel, W., & Müller, M. (1985). Proteolysis of mitochondria in reticulocytes during maturation is ubiquitin-dependent and is accompanied by a high rate of ATP hydrolysis. FEBS letters, 180(2), 249-52. Pubmed. []
  2. Margineantu, D. H., Emerson, C. B., Diaz, D., & Hockenbery, D. M. (2007). Hsp90 inhibition decreases mitochondrial protein turnover. PloS one, 2(10), e1066. doi: 10.1371/journal.pone.0001066. []
  3. Heo, J.-M., Livnat-Levanon, N., Taylor, E. B., Jones, K. T., Dephoure, N., Ring, J., et al. (2010). A stress-responsive system for mitochondrial protein degradation. Molecular cell, 40(3), 465-80. doi: 10.1016/j.molcel.2010.10.021. []

Submitted, home and with wheels

Jul 31st, 2010

About a week and a half ago I finally submitted my thesis, 40 months lab-work condensed into a document that was worryingly thinner than most. However, you work with what you have; padding the document with unnecessary figures and paragraphs will only serve to increase the length, to the detriment of the quality. As a secondary benefit, a short thesis also means that there is less to be familiar with for the viva, although I’m sure it will not work out that way in practice.

With the thesis submitted, there were few reasons to remain in Edinburgh; the viva itself isn’t until September. So last Saturday I headed south on the train, met my parents and a brother in Birmingham, and then headed off with my parents back to their house. For the first time in five years, I was back in my childhood room, not living out of a suitcase. It also meant, that for the first time in a year, I could get my desktop set up with a decent broadband connection again.

One of the problems with being back home, is the isolation. While I can’t pretend that my village is some rural backwater, without sewers, running water or electricity, it does lack basic facilities, such as a place to buy Jaffa cakes when you get a sudden craving at 8.30 pm.  This dire lack of Jaffa cake suppliers is made even more apparent when you consider the colour of my driving license: it’s green. The inability to drive may seem surprising for someone who grew up in a rural area, but despite the obvious usefulness of the skill, cars bore me, and early attempts at learning soon made it apparent that I wasn’t exactly a natural. Then it wasn’t long before I was off to university, and for the past eight years I have not only not needed a car, but have been living in situations where its often easier not using one. I’m sure I will learn eventually, but I’m wary of doing so, and then not driving for a few years.

Yet now I’m back in rural Wiltshire, and while I don’t intend to be here long, it is certainly far longer than I could cope with being housebound. On top of this, a somewhat lacking public transport system means that I’d be constantly relying on parents or other people for lifts, which is neither polite, fair, or convenient. This is especially true over the next two weeks, as my parents will be in Spain, and the drive back would require I give them a couple of days notice, hardly convenient; oh, and it might break the polite and fair requirements as well.

Without the ability to drive four wheels, I have instead settled on two, and have purchased my first bike for several years1; it has also been ten or more years since I last cycled. While they say that you never forget how to ride a bike, I was still a bit cautious as I set out on it for the first time. Last night I began with a simple ride up and down the road to make sure everything was flowing smoothly, and today I took it for a short 6 mile round trip to a local walking spot. Fortunately it appears that I do still remember how to ride a bike, and failed to have any embarrassing crashes. The trip also revealed that I need to raise my handlebars slightly, and to tilt the saddle back, something that will depend on my finding a suitable Alan key. The bike however does give me mobility, and in concert with the rail network it should mean I can get almost anywhere. And who knows, perhaps even if I do fail to drive a car for a few years after learning, I’ll slip back behind the wheel with similar ease to slipping back behind the handlebars.

I hope to get back to writing some of the more structured blog entries shortly, once I’ve had a bit more consideration about how I intend to use this website in future.

  1. A Trek 7.0 FX, if you are interested, or even for that matter, if you are not. []