Posts Tagged ‘ubiquitin’

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. []

Wikipedia Inaccuracies

Feb 10th, 2007

Oh dear. I like Wikipedia, its a lot of fun and usually reasonably accurate, but I just stumbled across an article which was almost entirely false. I can understand a few misconceptions sneaking in, but what possesses someone to write about a topic on which they clearly have no idea; especially when its something that specialist knowledge would seem a fairly obvious requirement.

The article in question was discussing the ubiquitin like protein Nedd8, something on which I’m currently doing a PhD. Ubiquitin is well characterised and has several roles, including targeting proteins to the proteasome, the ‘dustbin’ of the cell. Nedd8 is similar to Ubiquitin in structure, and the way it is used to tag other proteins. However it does not target proteins to the proteasome, and instead performs other functions. Yet the wikipedia article suggested that Nedd8 performed exactly the same role as ubiquitin, and accompanied this with ill formed sentences which gave little in the way of information and instead hinted at other severe misconceptions.

Neddylation is a protein degrading process analogous to ubiquitinylation in higher organisms (eukaryotes) in which NEDD8 is a key player. Unlike lysosomal degradation of proteins, a tagging occurs by attaching a small protein chain to mark for an enzyme complex (proteasome) by which they are then broken down. In fact, the term “neddylation” means a special form of ubiquitinylation initiated by an uncommon subset of proteins (see below), of which the initiators as well as the substrates are unique although the same pattern as in ubiquitinylation is followed.

So I have already addressed that Neddylation is not ‘a protein degrading process’ but furthermore neither is ubiquitination. The latter itself can result in protein degradation in some circumstances, but it itself is not protein degradation.

‘In which Nedd8 is a Key player.’ This badly formed sentence tells us little, and can serve to mislead. To suggest that Nedd8 is a key player in Neddylation is to suggest that bread is a ‘key player’ in a sandwich. Without the Nedd8 it simply isn’t Neddylation.

‘Unlike lysosomal degradation of proteins, a tagging occurs by attaching a small protein chain to mark for an enzyme complex (proteasome) by which they are then broken down.’ Surprisingly, if you can translate the sentence to English, this isn’t too objectionable; apart from the fact that it doesn’t belong here as Nedd8 doesn’t direct proteins to the proteasome. Furthermore as the sentence as it stands makes no sense, its possible that I’m just being charitable in my interpretation.

‘ In fact, the term “neddylation” means a special form of ubiquitinylation initiated by an uncommon subset of proteins (see below), of which the initiators as well as the substrates are unique although the same pattern as in ubiquitinylation is followed.’ A charitable reading could almost accept this, but its so garbled that such a reading is unlikely to occur to anyone not familiar with the subject. ‘A special form of ubiquitinylation’ is woolly at best. The process of Neddylation is analogous to that of ubiquitination (Or ubiquitylation, ubiquitinylation is a strange fusion of the two forms.*) and Nedd8 itself shows a great degree of homology (Similarity) to ubiquitin. But ultimately Neddylation is a ‘special form of ubiquitination’ in the same way a Renault is a special kind of Ford. It is misleading to say that the substrates are ‘unique’ as there are some targets which appear to be both Neddylated and Ubiquitinated in particular p53, however it would be true to say that their targets and target specificities are not identical. By initiators I will assume that the writer is referring to the enzyme cascade which leads to Neddylation. Again however they are incorrect, while the first two enzymes in the pathway, E1 and E2, are Nedd8 specific, no Nedd8 specific E3 ligases (the third enzyme) have been found, and it is suspected that the ubiquitin E3 ligases show dual specificity.

So ultimately there is only one idea in there which is remotely true, that ubiquitination and Neddylation are analogous processes. On reflection I get the impression that this is the only ‘fact’ the author knew.

I’ve pruned the article down to remove inaccuracies and will expand it later this week.

* Edited to add: Although a quick Google suggests this form isn’t completely unknown.

EDIT: I have closed this entry to comments as it was attracting a lot of spam.