Undelivered items go to BAG6

A story of how bin men work at the post office in our cells

Our cells produce all sorts of proteins with all sorts of functions. But to fulfil these functions the proteins must be in the right place. There’s no use for a protein that should be in the nucleus to stick around the outer membrane and like wise. When a protein needs to go to a specific compartment of the cell, this information is encoded in the protein itself. It’s like the address on an envelope. And interestingly, the machinery responsible for protein delivery starts working even before the synthesis of the protein is finished. It’s like we addressed an envelope, then started writing a letter, and before we finished it, our personal postman was already putting the address from the envelope into his sat nav.
Because there is so many various proteins being synthesised in the cell at any given moment, these postmen sometimes can’t keep up and happen miss a “protein letter”. The protein would then just stick around and clutter the wrong compartment of the cell. Now obviously, apart from post men, our cells are also equipped with bin men, who take care of such undelivered protein mail. A recent discovery (1) found that these bin men are actually very vigilant and work in a fashion very similar to the postmen. They hang around the protein factories, and as soon as they see a protein addressed to the membrane that wasn’t spotted by any of the postmen, they grab it and put it in their trash bag. Interestingly, one of the proteins responsible for this process is called BAG6.
You might wonder, is this discovery really that important? Isn’t it science just for the sake of it? Would a cell really mind having some litter laying around its protein synthesis offices? Well, actually, it would. Apart from just not being able to fulfil its function at the right compartment of the cell, a protein that isn’t where it’s supposed to be, can behave in an unpredicted and sometimes harmful way. This is one of the causes for Creutzfeldt-Jakob disease, otherwise known as the human form of mad cow disease. A so-called prion protein, the cause of this disease, is normally supposed to find itself in the cell membrane. But when it doesn’t and stays in the cell cytoplasm (where protein synthesis occurs) it tends to form aggregates which are very resistant to being cleaned up. These aggregates start affecting the functioning of the whole cell, and because the protein is mainly produced in the brain cells, the brain’s function gets severely impaired. If this research goes further, maybe the scientists will be able to better understand the onset of the disease, and maybe even come up with novel therapies to either treat patients affected by it, or prevent its development in people who have a family history.
So that’s the story. In our cells, bin men work as fast as they can to remove junk proteins which are undelivered. And wouldn’t it be great if all the junk mail we receive was binned just as it’s being put in its envelope?

1. Protein targeting and degradation are coupled for elimination of mislocalized proteins.

Trans-differentiation: cells in trance do things they haven’t dreamt of

Our bodies get injured, get damaged, get old and we’d like to be able to regenerate them. Modern medicine has for quite some time been on it’s way to help us with this. It’s all about the stem cells! The magnificent almighty stem cells that can turn into any adult cells that need replacing. The road to actually achieving this magical regeneration has been quite bumpy and riddled with ethical issues, because embryonic stem cells – which have the biggest differentiation potential – are generated from undeveloped human embryos. But with the whole idea of “manufacturing” cells for regeneration – have we got the wrong end of the stick from the beginning? Recent findings suggest we could have took a much more straightforward approach. But only if we had dismissed what our professors told us when we were students and done some procedures in spite of them saying they had no chance of working. And seen them actually work in the end.

Embryonic stem (ES) cells obtained from many species, including human, have been around for quite some time (1, 2). We’ve seen them differentiate into neurons, heart muscle cells, liver cells and so on . We’ve also seen them raise serious ethical issues of whether obtaining them from very early-stage embryos is killing or not. We’ve seen them cause a see-saw changes in US law imposing and lifting bans on ES cell research. So the scientists had a look at another kind of stem cells – the adult stem cells. These can be obtained from an adult person without killing them, so there was no strong ethical objections against these. But many researchers argued these cells had very limited potential of differentiation. Our body organs develop from one of three layers of an embryo – the outer, the inner, and the one in between. Some scientists strongly advocated the idea that when you isolate adult stem cells from, say, bone marrow – which comes from the middle layer – they will never be able to give rise to neurons (originating from the outer layer) or liver cells (the inner). So the bottom line was, adult stem cells – ethical, but not so good.

Then came the induced pluripotent stem cells, or iPS cells (about which I wrote some time ago). They are generated from adult tissues, as easily accessible as skin, but they behave like embryonic stem cells, i.e. can readily differentiate into all kinds of cells from any embryonic layer. It was a big wow in the scientific society, as these cells were as plastic as the unethical embryonic stem cells, yet as ethical as the crappy adult stem cells. Win-win? Not exactly. One practical issue scientists experience with both ES and iPS cells is that we can never achieve full differentiation of all the cells while they’re in culture. Say, you have a patient who suffered a bad liver damage so you need to inject them with liver cells. You can culture the ES or iPS cells, differentiate them into liver cells and then inject them into that patient. But the few cells that failed to differentiate in culture, will start doing it after being injected. And the problem is – they can go crazy and out of control in the process. They can differentiate into weird tumour masses called teratomas, which can contain any sort of tissue. Some teratomas were even found to contain hair or teeth. Now, you don’t want that in your liver, do you? Neither do the doctors who treat you. This is the main reason why both ES and iPS cells, even though we’ve been culturing the former ones for about three decades now, have been struggling to be actually accepted for clinical trials.

Recently, there is a new wave of cell differentiation research, which stays away from stem cells altogether and is in opposition to what they taught us at our university courses. They used to tell us that once a stem cell differentiates into a specialised cell – that’s it! There’s no becoming a another kind of cell. Or in scientist’s words – there’s no trans-differentiation. This is in line with the argument that adult stem cells from one embryonic layer can’t differentiate into cells from another. However, recently there are more and more scientific reports showing how not only adult stem cells can trans-differentiate into other-layer cells, but adult specialised non-stem cells can do that too. Apparently, you can take a skin cell, and genetically reprogram it to become a neuron cell (3)! That’s very exciting, but skin and neurons actually come from the same embryo layer – the outer. But other researcher have broken this boundary, and transformed fibroblasts (the middle) into liver cells (the inner, 4). And even more importantly, the newly generated liver cells were shown to be working in living organisms. For instance when the skin-turned-liver cells were injected into mice with liver damage, they took part in reconstituting the organ tissue. No embryo killing so it’s ethical. It can be done without changing the adult cells into the iPS cells first. No iPS cells, no risk of teratomas. Win-win? Hopefully!

The research is still in early stages and a lot more testing needs to be done. Also, because of a short life span of a mouse, we can’t really predict how would these cells behave, say, ten years after injection. Would they still be functional? Would they form some kind of tumours or cancers? We can’t exclude these possibilities yet. But you know what we scientists are like. We will keep trying until we get the answer. And hopefully the answer will be – the trans-differentiated cells are long lasting and safe.

1. Establishment in culture of pluripotential cells from mouse embryos
2. Embryonic Stem Cell Lines Derived from Human Blastocysts
3. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts
4. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors

An immune system protein helps ovarian cancer survive

Scientists have discovered that a well-known protein, when produced by ovarian cancer cells, protects them from being killed by the immune system. This might apply to other kinds of cancer as well, opening doors to new immunotherapies and hopefully helping in the fight against cancer.

What makes us distinct from one another, from an immunological point of view, is a group of genes called HLA. They are varied across the human population and it is these genes that need to be tested and perfectly matched when looking for a transplant donor. One of them, called HLA-E, seems to also have a role in cancer, and this role is good for the cancer, bad for the patient. Marloes Gooden and colleagues made this discovery in ovarian cancers (1), which are usually diagnosed quite late and have poor prognosis, as only about one in two diagnosed women will live longer than 5 years.

Any cancer that arises in our body can be recognised and attacked by the immune system. In turn, the cancer cells can develop some strategies to evade the immune response and HLA-E production seems to be one of these strategies. Cancer masses are often infiltrated by immune system cells called cytotoxic T lymphocytes, or CTLs. They act a little bit like very aggressive policemen who chase and kill criminals. CTLs have the natural ability to recognise and kill any cell that doesn’t appear normal, which in this case are cervical cancer cells. But when a cancer cell produces this HLA-E protein, it seems to be functioning as an anti-CTL taser. The CTL is still there, in the tumour, and even though it might be able to “see” the nasty cancer cell, HLA-E makes it not do nothing about it.

The researchers analysed ovarian cancer samples from hundreds of patients, many of whom already died. When they looked at the amount of CTLs in the samples it became pretty evident that the more CTLs infiltrate the cancer (the more policemen chase the criminals), the longer the patient will live. But the picture didn’t seem quite complete at that stage. They decided to divide the cancer samples into two groups: one where the HLA-E levels were very low or even undetectable, and one where they were high or very high. In the first group they made a similar finding: the more CTLs infiltrated the cancer mass the longer the patient lived, and the difference in survival was even more evident. But when they looked at the patients with high HLA-E levels in the cancer cells, it turned out that the number of infiltrating CTLs did not make any difference to how long the patients would live.

The authors do mention that they stumbled upon more molecules that might be connected with this whole phenomenon, but just discovering this important function for HLA-E is an important piece of the puzzle. There are currently quite a lot of established and experimental therapies for cancer called monoclonal antibody therapies. They way they work, is by giving the patient antibodies against molecules that seem to facilitate cancer survival and aggressiveness. The study mentioned here might have identified one of these molecules and hopefully, in the future, new anti-HLA-E therapies will let women with ovarian cancer live much longer.

1. HLA-E expression by gynecological cancers restrains tumor-infiltrating CD8+ T lymphocytes.

Stepping over the stop

Imagine you have a robot that is baking a cake according to a precisely written recipe. And when it finishes making the cake base, the recipe says “stop here”, even though there are instructions for a creamy topic below. Cells do a similar thing: in an RNA molecule they have a recipe for a protein molecule, and they follow the instructions exactly, until they reach a ‘stop’ word. A recent finding by Karijolich and Yu (1) has shown that, at least in yeast cells, there are enzymes that can change the “stop” instruction and make the cells read it as continuation of the protein and keep reading the instructions further. It’s as if you erased the “stop here” instruction from the recipe and gave it back to the robot. After making the base it would go straight to making the topping.
Why would the cells do that? Sometimes they might need the protein to be bigger, because the extra bit can provide extra functions. For instance it can cope better in stressful conditions. In normal circumstances, introduction of such an extra bit would be a waste of energy. But when the environment changes from normal to stressful, the cells don’t need to produce a whole range of new protein-coding RNAs, they already have them, and all they need to do is change “stop” to “go on” and voila! A new protein that copes well with environmental stress. And it needn’t be stress, there can be plenty other things that can change inside or outside of the cell, and having such RNA molecules is quite a fast way of responding to these changes. It’s like you just found out that your friends are bringing kids to the dinner party and the kids just loooove creamy topping. You don’t need to prepare a whole new set of instruction for your robot to follow, you just remove the “end here” bit and a cake with creamy topping is on the way.
In the publication I’m mentioning here, the experiments were done in yeast. But it’s a well known fact that plenty of the processes that go on in yeast cell also happen in higher organisms – including humans. What implications has it got? In this publication, the cells were discovered to edit the “stop” instruction in the RNA molecule, but it is very likely that other bits of the instruction might be altered as well. This might lead to substantial change in how we understand the relation between instructions in RNA and what protein we expect to be made from these instructions.

1. Converting nonsense codons into sense codons by targeted pseudouridylation

How anti-genes can make diabetes worse.

Type 2 diabetes was recently declared an epidemic, as its incidence doubled from 1990 to 2005. Scientists have found several new genes that seem to be contributing to the condition and are now hoping this might be the new target of anti-diabetic drugs.

Sugar that we consume with our food gets from our gut to our bloodstream and then from our bloodstream to our body cells. The latter process is regulated by insulin. Sugar can enter the cells through a special door that are normally locked. Insulin is the key, and the lock on the cell’s door is called a receptor. Diabetes can result from either our body not producing insulin so that there’s no key to open the door (which happens in type 1 diabetes) or the locks in the doors get messed up in all cells so no matter how much keys you try – you can’t open the doors (which results in type 2 diabetes). Why the locks get messed up in our body cells is not exactly clear. The recent finding, however, sheds some light on how several genes might be contributing to this.

Scientists concentrated on two genes – miR-103 and miR-107. They aren’t regular genes that encode proteins, they are called micro RNA – small bits of RNA that can stop production of other proteins. They are – in a way – anti-protein genes. Trajkovski and colleagues(1) did two things with these genes in mice tissues – either over-activated them in healthy animals or silenced them in ones that suffer from type 2 diabetes. What seems pretty clear from their results is that the more active these genes are the more diabetes 2-like symptoms the animals showed. And like wise – silencing these genes in diabetic animals reduced the severity of their symptoms. The researchers even went a step further and asked why this might be. As I mentioned, the two genes they focused on do not code proteins but actually can stop the production of some of them. And that was the case here as well – the two genes prevented generation of caveolin 1, a protein which can be thought of as an important element of the lock in the door opened by insulin.

Scientists are obviously excited about this as this opens a whole new venue for treatment of type 2 diabetes. And what is also very exciting for me personally, is that when they set off to find new genes involved in diabetes, they found 5 of those non protein-coding micro RNAs. To keep things simple they only analysed two of them, but even with those two they got significant results. I’m looking forward to some more research on the other three.

1. MicroRNAs 103 and 107 regulate insulin sensitivity

Are even better vaccines on the hoRNAzon?

To vaccinate our bodies against lethal diseases, like smallpox, we need to have the pathogen injected into our bodies in order to teach our immune system how to recognise it and fight it. Obviously we cannot use a perfectly healthy microbe for this injection as this would give a result quite opposite to intention. Therefore, many vaccines are made of bacteria or viruses that were killed prior to injection. Killed pathogens can not do any harm to our bodies, but are good enough to teach our immune system what they look like, so that should their healthy counterparts infected us in the future, our white blood cells are fully equipped to fend them off almost immediately. But for some vaccines the immune response isn’t quite as strong as during an actual infection with the living version of the bug.
Leif Sander and colleagues went for a hunt for what it is about living bacteria that makes them more irritating to our immune system. To their (and mine) great surprise the part of bacteria that seems to be doing a big part of the job, was RNA, a kind of molecule present in all living cells. Firstly, they analysed killed bacteria for several kinds of life-building blocks (including DNA and RNA) and found that RNA was the only one that was rapidly destroyed when bacteria were being killed. Their subsequent experiments were quite straightforward – they treated immune cells with either living bacteria, dead bacteria, or dead bacteria mixed with intact bacterial RNA, and measured how aggressively immune cells would react to these treatments. They also used the three mixtures to inject living animals to see how they would response to such vaccinations. In both cases – immune cells cultured in vtiro, and in living animals – the immune response was strong for living bacteria, weaker for dead bacteria, and strong again (or sometimes even stronger) for dead bacteria mixed with bacterial RNA.
Obviously this is the first observation of such kind, but the potential implications of it might be really beneficial. Often vaccines don’t have a 100% success rate, i.e. not all vaccinated people are protected from the actual disease. If in future, we’d be able to develop vaccines which would contain a mix of dead bugs with their intact RNA, we might be able to provide much more successful protection of the population. Watch this field! I certainly will.

1. Detection of prokaryotic mRNA signifies microbial viability and promotes immunity

Don’t miss! Totally ethical embryonic stem cells! Now 100 times more for 4 times less!

This news was published already on April 8^th this year, but I have only recently come across it.

Induced pluripotent stem (iPS) cells have been a very sexy topic since they were first generated in 2006. They are very much like embryonic stem cells, but their generation involves no embryo killing and thus they completely circumvent the whole ethical debate. To generate these cells, in a nutshell, scientists took some fibroblasts (adult cells) and treated them with retroviral vectors (a sort of molecular syringes) which carried genes that can make a cell go into embryonic – or pluripotent – state. This way they reprogrammed the fibroblasts to return to embryonic-like state. But the efficiency of this procedure was extremely low, as only about 0.02% of human fibroblasts turned into iPS cells (1). And when you think about it, it’s not much of a surprise. To reprogram a cell, you need three or four genes, which is three or four separate viral vectors. If a viral vector infects only, say, 20%, of a cell population, then two vectors will infect only 20% of that 20%, and so on. On top of that, not all infected cells will give in to the effect of the introduced gene. In the end, you end up with only a small fraction of cells that both took up all the genes and underwent successful reprogramming.

In the publication that I am going to comment on here (2), the scientists took a slightly different approach. They also used the retroviral vector as a gene delivery tool. However, instead of four, there were able to use only one retroviral vector. Normally, it’s hard to fit many genes into one vector, but these guys used only two – not three or four – genes. And on top of that, they were a very different kind of genes coding so-called miRNA. Now, unlike other genes, miRNAs don’t get translated into proteins but they can be very powerful when it comes to regulating levels of other proteins. And on top of that – miRNA genes are far smaller than protein genes, so it was not so much of a challenge to fit two of them into one vector. Effectively, using this technique they were able to generate iPS cells with about 100-fold higher efficiency!

This is pretty exciting but still pretty recent. If it is going to repeated by other researchers, chances are, this will become the standard protocol for generation of iPS cells. Which in turn hold a great promise for serving as good in vitro models of genetic disorders and maybe – in future – for personalised regenerative medicine.

1. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. 
2. Highly Efficient miRNA-Mediated Reprogramming of Mouse and Human Somatic Cells to Pluripotency.