The cell's ironing: protein folding and folding surveillance

Proteins are highly organised structures: they're folded into particular conformations;associated with other protein subunits and have different levels of organisation (see previous post).

But how do they achieve their folded state?

(1) How do proteins fold?

We all like help-so do proteins

Sometimes it is easier when someone helps you fold away your clothes. Same for proteins-yes they are largely self-folding. But what happens when this goes wrong?

Chaperones are part of the cell’s emergency services attending various accidents (misfolded proteins) but in a disaster they all flood to the scene (when a cell is under stress).Many chaperones are heat shock proteins (Hsp) and are highly abundant when a cell is under stress (during nutrient starvation, extreme temperatures, exposure to toxins etc).

Aggregates can easily form when hydrophobic regions of the protein can end up on the outer surface of the misfolded or partially folded protein and will attract other misfolded proteins with exposed hydrophobic residues (as the cell has an aqueous environment the water fearing residues want to bury themselves in the core of a protein).  In a living cell it not just empty space (imagine trying to fold your washing in a crowd without elbowing anyone!). Chaperones help the protein fold in the most energetically favorable way as well as preventing the unwanted binding of other proteins during the folding process.

Some molecular chaperones (Chaperonins-Hsp60) act like a huge tent in the middle of the crowd where you can fold your washing without people getting in the way. They completely isolate the protein from the environment of the cytosol.

(2)Bacterial  Hsp60 (GroEL) and associates Hsp10 (GroES)

Whilst other chaperones (Hsp70) bind to a region of the protein to aid folding like having a group of people who are designated to aid in folding of washing-it is their job.

(3) Hsp70 binds to hydrophobic regions to aid folding

Chaperones are not just involved in folding they’re also important for unfolding, refolding and trafficking.  Chaperones work by an ATP* dependent mechanism: they bind to hydrophobic regions of a misfolded (non-native) protein.

One size of chaperone doesn't fit all:
There are different classes of molecular chaperones (they are usually classified by their molecular weight).  Hsp60 (chaperonins) ,Hsp70,Hsp90 and Hsp100 systems. The numbers indicate the molecular mass of each Hsp protein subunit.

Focusing on the chaperones involved in protein folding:  namely- Hsp60,70 and 90.
They generally act under two systems Hsp70 and Hsp60 (chaperonins).

Hsp70 interacts with newly synthesised proteins and releases them . Whilst chaperonins  (Hsp60) are large cylindrical protein complexes ( figure 3) (composed of several polypeptide chains-quarternary structure: an outfit (see previous post)).They enclose the proteins for folding. The two systems act sequentially Hsp70 act on newly synthesised proteins and then chaperonins assist folding of proteins that do not reach their correct folded state by Hsp70 alone.

So imagine you (Hsp70)are folding your washing and your Mum (Hsp60) is stood by your side and when something is difficult to fold or you have not folded it correctly your Mum takes over to ensure correct folding.

The Hsp90 system functions downstream of Hsp60. It acts in the late folding stages of proteins involved in cellular signalling and development. Both Hsp70 and Hsp90 can direct proteins for degradation.

It is now like your Grandma (Hsp90) has joined the folding party; smoothing out the creases of odd item that your Mum (Hsp60) has missed.

Folding avoids creases but what does it mean for proteins?

When proteins fold they gain a particular shape which is important for the function of that protein. It may allow it to associate with other proteins to form a complex.

It has been suggested that the decline in the cell’s ability to correctly fold proteins and maintain correct protein folding (proteostasis) declines with age. This can allow the formation of protein aggregates which are involved in many neurodegenerative diseases such as Alzheimer’s and Parkinsons disease. Finding out more about chaperone mediated surveillance of proteins is vital as these diseases become more prevalent in our aging society.

(4) Comparison of a healthy neuron with a Alzheimer's neuron with protein aggregates ( Beta amyloid plaques and neurofibrillary tangles)

*ATP= Adenosine triphosphate is the energy currency of the cell. It is a phosphorylated nucleotide that is made and consumed by all cells and drives chemical reactions

References

Hartl F, Bracher A, Hayer-Hartl M., 2011. Molecular chaperones in protein folding and proteostasis., Nature reviews., 475., pp324-332

Lodish H et. Al., 2008., 6^th Ed., Molecular cell biology., W.H.Freeman., New York

Saibil H., 2013. Chaperone machines for protein folding, unfolding and disaggregation., Nature reviews., 14., pp631-632

Images

(1) Left: https://thelaundrycenter.com/blog/tag/wash-and-fold-laundry-service/

      Right: http://ocw.mit.edu/courses/biology/7-343-protein-folding-misfolding-and-human-disease-fall-2004/

(2) Left: http://www.terra-nova.co.uk/tents-and-spares/tent-inner/

     Right: http://www.tulane.edu/~biochem/med/groel.gif

(3) Top: http://www.singaporelaundry.com/articles/how-to-fold-clothes.html

      Bottom: http://www.pnas.org/content/103/16/6166/F1.expansion.html

(4) https://sites.google.com/site/bme365ralzheimers/pathophysiology

If proteins were in my wardrobe: folding and organisation

So my blog has been very quiet (apologies). A new start to the academic year so a new blog post!


When you open your wardrobe, how does it look?

Like this?

(1) Clean and tidy

...Or admittedly more like this?

(2) Disorganized and messy

Many things in molecular cell biology can appear rather chaotic on the surface. Proteins look like highly higgledy piggledy molecules. When  actually they’re pretty organised things: structurally complex and sophisticated molecules of life.

How the cell folds its wardrobe

Amino acid sequence: The shape of a protein is determined by its amino acid sequence (primary structure)- the threads of the garments (20 types in total). They can be grouped into different categories according to their side chains-acidic, basic, uncharged polar and nonpolar.

(3) 20 Amino acids 

Non covalent bonds also influence the folding of proteins: hydrogen bonds, ionic bonds and Van der Waals attractions.

Environment: The cell has a very aqueous environment which affects a proteins shape, making it very compact. The non polar side chains are hydrophobic (water-fearing) bury themselves in the core of the proteins whilst the polar (hydrophilic- water-loving) side chains interact with water and so tend to gather on the outside of the protein.

When completing a task we often look for the easiest way to accomplish the task with minimal cost (whatever that may be) Proteins do the same: they fold into a shape of the lowest energy (i.e energetically favourable to maintain- the easiest way to fold).

Organisation of protein structure

Unlike my wardrobe proteins have a very organised structure that can be broken down into four categories:

(4) Categories of protein structure 

Many proteins have large structures (of one very long string on amino acids folded up) which gives a further unit of organisation: the protein domain.

 This is a length of the protein which is between 40 and 350 amino acids (stitches) in length. This length folds independently to the other regions of the protein like different parts of a shirt (collar, sleeve,  the pocket) are all still part of one shirt but and useless on their own yet still independent.

Different protein domains often are associated with different functions.

Items in a cells wardrobe (some examples)

Src protein kinase:  One of the many secretaries of the cell. It acts in signalling cascades by adding phosphate groups from high energy donor molecules like ATP to substrate molecules.
A protein formed of four different domains (each has a different function).

The SH2 and SH3 domains are involved in regulation whilst the catalytic domain and the activation loop play a role in the catalytic activity of the kinase.

(5) Src Kinase Hck

Haemoglobin: Found in red blood cells and vital for the transport of oxygen around the body and also has roles in the transport of carbon dioxide and hydrogen ions. It is a multi-subunit protein: two α subunits (two protein chains of 141 amino acids-stitches) and two β subunit (two protein chains of 146 amino acids-stitches).

(6) Haemoglobin

So next time you open your wardrobe to put your clean clothes away (freshly synthesized (nascent) proteins) think about how organised our cells are: folding proteins and packaging them to be sent to various locations within the cell. How organised is your wardrobe?

Upcoming posts
More about protein organisation and folding-why and the cellular machinery involved
What happens when folding goes wrong?

References

Alberts B et al., 2008., 5^th Ed., Molecular biology of the cell., New York., Garland Science

Berg  J et al., 2011., 7^th Ed., Biochemistry., New York, W. H. Freeman

Images

(1)  (https://www.flickr.com/photos/8011986@N02/4211768335/)

(2) (http://imgarcade.com/1/messy-bedroom/)

(3) (http://www.personal.psu.edu/staff/m/b/mbt102/bisci4online/chemistry/chemistry8.htm)

(4) (Adapted from: http://en.wikipedia.org/wiki/Protein_structure_prediction)

(5) (Adapted from: http://mysbfiles.stonybrook.edu/~tomiller/dualrole.htm)

(6) (Adapted from: http://www.rcsb.org/pdb/general_information/news_publications/newsletters/2004q4/education_corner.html)

I'm sorry, bio-what??!!-A brief plug for biochemistry

University is a time where you meet lots of new people. Being in a new city and getting involved in a new community. You get asked the three questions a zillion times:

1)What's your name?

2)Where are you from?

3)What are you studying?



A tip for you all!

 

Okay so I study biochemistry. But I thought I'd give an insight into how diverse a subject it is.

Biochemistry is not quite a fusion of your biology and chemistry lessons you might've had at school
It is much more than that!

Biochemistry is at the heart of many areas of the life sciences such as genetics, cell biology, energy and metabolism, plant biology and development of disease.

Lubert Stryer, the famous biochemist and author of Biochemistry (W.H. Freeman & Co.), states that biochemistry is “rapidly progressing from a science performed almost entirely at the laboratory bench to one that may be explored through computers. Its practical approach applies the molecular aspects of chemistry to the vast variety of biological systems."

 The thing I love about studying biochemistry is taking a 'simple' cell and finding out the intricate mechanisms that enable that cell to function. For example protein synthesis: DNA is transcribed into messenger RNA (ribonucleic acid, a molecule similar to a single strand of DNA). The code of bases that the mRNA contains is transcribed by ribosomes into a chain of amino acids joined by peptide bonds a.k.a a protein.

That may sound simple summarised in just a few short sentences.But it's far from it!  And that is only one example!

Molecular biology shows that there is to life that meets the eye which makes me feel privileged to be studying biochemistry.

Upcoming posts
Sugar and sweetners-What's the difference?

The gut feeling about caffeine

 Following from my previous caffeine post: What are the effects of caffeine on our bodies? I thought it's time to round off my investigation into the intriguing substance of caffeine.

Not the best way to find out the effects of caffeine on the body

Do you ever get that gut feeling when drinking coffee?-Coffee and the gastrointestinal system

There is strong evidence that coffee increases gastric acid secretion. Interestingly experiments have shown that it's the other constituents in coffee that contribute to this increase in gastric acid secretion.
This was shown by  measuring the dose response of caffeine, regular coffee and decaffeinated coffee for gastric acid secretion in normal subjects. Both regular coffee and decaffeinated coffee gave a similar response in gastric acid secretion which was higher than that of caffeine alone (on a cup equivalent basis) (Cohen and Booth, 1975).

The dose response? what??

Measuring the dose-response enables scientists to observe the change in an effect (in this case:gastric acid secretion) caused by varying the levels of dose of a substance (in this case: dosage of caffeine, regular coffee and decaffeinated) after a certain length of time.

Kidney function

The kidneys play an important role in filtering the blood. The kidneys remove waste products (such as urea) and extra water from the blood which form urine. Any drinker of caffeine beverages is well aware that caffeine tends to stimulate an increased flow of urine.

Anatomy of Kidney

Caffeine was traditionally used to increase urine output until more potent diuretics became available. The diuretic effects of caffeine appear to be due to an increased rate in blood flow to the kidneys and increased rate of blood filtration. These affects are due to antagonism of circulating adenosine (see previous post) having a regulatory role in the formation of urine  (Fredholm 1984).

So will my cup of coffee cause a fluid imbalance?
A dose of 300mg of caffeine (approximately 4-5 cups) can cause acute diuresis- this has been shown by several studies (Oswald and Schnermann., 2011). Caffeine will only cause a significant increases in the volume of urine excretion and a negative fluid imbalance in a large dose. A study in which caffeine was given (6mg/kg) for 11 days showed no effect in daily urine volume (Armstrong et.al 2005).

Time for a breather-Respiratory system

Caffeine is a respiratory stimulant (Braun 1996). However based on the average person's caffeine consumption has little effect on the respiratory system. Larger doses of caffeine has proven to be effective in the treatment of neonatal apnea-the cessation of breathing in newborns.

Upcoming posts

How about  I keep it a surprise this time?

References

Armstrong LE, Pumerantz AC, Roti MW, Judelson DA, Watson G, Dias JC, Sokmen B, Casa DJ, Maresh CM, Lieberman H, Kellogg M. 2005., Fluid, electrolyte, and renal indices of hydration during 11 days of controlled caffeine consumption. Int J Nutr Exerc Metab. 15

Braun S., 1996., Buzz: The Science and Lore of Alcohol and Caffeine., Cary., NC., USA., Oxford University Press
., 252-265. Cohen, S., and Booth, G. H. 1975., Gastric acid secretion and lower-esophageal-sphincter pressure in response to coffee and caffeine., New England Journal of Medicine., 293,897-899.

Fredholm, B. B., 1984.,Cardiovascular and renal actions of methylxanthines., New York:Alan R.Liss

Oswald H and Schnermann J., 2011., Methylxanthines and the Kidney., Handbook of experimental pharmacology., 200., 391-412.

Why can't the traffic on our roads be more like the traffic in cells?

Time to take a short break from caffeine. After all it's a special occasion with the announcement of the Nobel Prize 2013 winners! (It's about time I did a post about it)

I see science being a bit like a mystery to be solved: all the clues have to be gathered together to see the overall picture. However like scooby doo and his gang it is much easier solving the mystery together!

Likewise this years noble prize in medicine was awarded to three scientists: Randy Schekman, James Rothman and Thomas Südhof. Who uncovered how the cell organizes the transport of substances around the cell.

Cells have traffic?

So you're probably thinking how do cells have traffic?

Rules of the road: Cell style

Substances are transported around the cell in membrane bubbles called vesicles (these are a bit like taxis transporting passengers from A to B). Vesicles bud and pinch-off from specialised structures in cells called organelles. Vesicles travel along the cytoskeleton of the cell like taxis travel along roads. The cytoskeleton of the cell is a network of protein filaments and tubules in the cytoplasm of many cells. As well as its role in transport the cytoskeleton gives the cell shape.

Red showing cells actin cytoskeleton

Randy Schekman's contribution

He discovered a set of genes that were required for vesicle transport.
How did he do this?
He studied the genetic basis of how a cell organises its transport system by using yeast as a model. (Yeast  are often used experimentally as they can be grown quickly and have a eukaryotic cell structure-they have membrane bound organelles like human cells, we also share many genes with yeast cells). Sometimes the best way to discover how something works is to look at what happens when that system goes wrong. That's exactly what Schekman did. He studied yeast with defective transport systems, identified the cause of congestions and identified the mutated genes. So he had solved one aspect of cellular transport.

James Rothman's contribution

He revealed the proteins involved in the fusing of vesicles (the transporters of cellular cargo)  with their targets (their desired destination in the cell) enabling the vesicles to deliver their cargo. Through his experiments he showed that these proteins bind in specific combinations to ensure that cargo is delivered to a specific location.

Thomas Südhof

He was interested in how nerve cells communicate with each other in the brain. He showed how vesicular contents can be released as a result of a cell signal.

In nerve cells signals come in the form of neurotransmitters (chemicals such as acetylcholine and noradrenaline). Nerve signals pass from nerve cell to nerve cell at junctions called synapses. Südhof found how nerve cells release neurotransmitter into the synaptic cleft upon a signal.

When an action potential (a electrical signal) arrives at a nerve terminal, calcium ions enter the cell through a channel that is temporarily open due to the arrival of the action potential. The entry of calcium  causes vesicles containing neurotransmitters to be released into the synaptic cleft where the signal is communicated to the neighbouring nerve cell. Südhof discovered the calcium sensing proteins in the pre-synaptic nerve terminal which direct and dock vesicles containing neurotransmitter to the cell membrane where the neurotransmitter is released into the synapse (using the mechanisms discovered by Schekman and Rothman). See home-made diagram below (apologies I am no artist).

Synapse- Signalling between two nerve cells

Cells are incredible at transporting substances. It's interesting to see how the work of a few scientists have uncovered the mechanism of transport of substances. Hats off to them! I hope next time you're travelling you'll think about the trillions of cells in your body transporting substances to and fro.

Upcoming posts
More posts on caffeine are inevitable. Will hopefully be looking into reviewing scientific papers in the future-Watch this space.

What are the effects of caffeine on our bodies?

An advertisement in a London newspaper in 1657 described coffee as: "A very wholesome and physical drink that helpeth digestion, quickeneth the spirits and maketh the heart lightsom, is good against eye-sores,coughs, colds, rhumes, dropsie, gout and scurvy." (Braun S., 1996)

Obviously today we do not view coffee and caffeinated substances to be this marvellous medical elixir.

So what are the effects of caffeine on the different systems in our bodies?

I'll be starting with the nervous and cardiovascular systems

Nervous and cardiovascular systems

Caffeine and the nervous system

Caffeine is a central nervous system stimulant-It increases brain and spinal cord activity, speeding up physical and mental processes.

Caffeine also affects the autonomic nervous system -A branch of the peripheral nervous system which controls smooth muscle, internal organs and glands (click for more information on the nervous system).
There has been reports that caffeine increases skin conductance (sweat gland activity). A 500 mg anhydrous (dry weight) caffeine dose can produce behaviour similar to General Anxiety Disorder also referred to as 'caffeinism' (Bruce et al., 1986). Caffeinism is a condition resulting from ingestion of a large dose of caffeine shown by diarrhoea, elevated blood pressure, restlessness and insomnia.

Putting things into perspective: a 500 mg dose all at once is a large amount considering  an average cup of instant (heaped tea spoon) coffee contains 60 mg (Center for science in the public interest., 2012). A large dosage of caffeine can produce extreme symptoms due to the rapid rate at which  caffeine is absorbed. So do not fear drinking your morning cup of coffee as 500 mg is a high dose and was used for experimental purposes.

Note how some of the effects caffeine has on the nervous system appear anxiety reducing (i.e increasing concentration and reducing tiredness) and some effects similar to anxiety (insomnia and restlessness).

Cardiovascular system

 Caffeine has an interesting effect on blood flow. In moderate amounts caffeine can increase blood pressure-the effect is more significant in those who do not regularly consume caffeine (Bruce., 1986: James 1997). But surprisingly has little effect on heart rate (France and Ditto 1992).

How does this change in blood flow come about?
Caffeine molecules compete with adenosine for adensosine receptors on cardiovascular cells (See previous post on receptors). In the cardiovascular system adenosine causes dilatation of coronary and cerebral blood vessels. Caffeine inhibits this process, meaning the blood vessels remain constricted. The narrower diameter of the blood vessels result in higher blood pressure (James 1997).

Time for a little analogy......
Imagine you have gone shopping for a new garden hose. Your standing in the aisle looking at the different diameters of garden hoses you can buy. Whilst you do this you imagine watering your garden with your new hose. The pressure of the stream of water coming out of the end of the hose depends on the diameter of the hose. Logically the narrower the diameter of hose the higher the pressure.

Hoses varying in diameter

Like hoses our blood vessels are the same. The narrower the vessel the higher the blood pressure in that vessel. But blood vessels (unlike your garden hose) are clever and can change diameter.

When adenosine is present in the blood, this causes the smooth muscle in the blood vessels to relax- the vessel wall gets thinner and the space where blood flows in the vessel gets wider: this is vasodilatation. When caffeine is present and binds to adenosine receptors instead of adenosine; it has the opposite effect. The smooth muscle in the vessel wall contracts and gets thicker so the space where blood flows gets narrower resulting in a higher pressure in that vessel: this is vasoconstriction.

Upcoming posts....

I'll be looking at the physiological effects of caffeine on the gastrointestinal and respiratory systems and also the effect on renal function (think peeing!). I also hope to do a bit about caffeine and health.

References

Braun S., 1996., Buzz: The Science and Lore of Alcohol and Caffeine., Cary., NC., USA., Oxford University Press

Bruce M, Scott N, Lader M, Marks V., 1986., The psychopharmacoloical and electrophysiological effects of a single dose of caffeine in healthy human subjects., British Journal of Clinical Pharmacology., 22, 81-87., [Online]., Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1401080/?page=1 ., [Accessed: 02/10/2013].

Center for science in the public interest., 2012., Caffeine content of food & drugs (online)., (Updated 2012)., Available at: http://www.cspinet.org/new/cafchart.htm., [Accessed: 03/10/2013].

France C and Ditto., 1992., cardiovascular reponses to the combination of caffeine and mental arithmetic, cold pressor and static exercise stressors., Phychophysiology., 29., 272-282.

James J., 1997., Understanding Caffeine., California., USA., SAGE Publications Inc

Puzzling over caffeine and adenosine-it's time to talk about receptors (please don't be antagonistic!)

Heading back to university for the start of the academic year it'll be inevitable that my coffee consumption will increase exponentially. So I'm back again and thinking about what happens in my body when I consume my daily caffeine fix.

After looking at caffeine metabolism and whether caffeine is addictive and causes insomnia in my previous posts. I thought it was time to start looking at HOW caffeine changes our body's functioning.

So time to start fitting the pieces together of the physiological effects of caffeine. This starts with receptors.

What are receptors?

Receptors are proteins which can be found on cell membranes. They bind to specific chemicals (these could be hormones, drugs, neurotransmitters etc). The binding of these chemicals can cause a change to occur in the cell.

It is natural when looking at the biological effect of caffeine in the body to start looking at what happens when caffeine binds to a receptor.

The caffeine-adenosine relationship

Everybody has two friends which are uncannily similar to each other. Caffeine and adenosine are those uncannily similar friends-they have a similar molecular structure.

Spot the difference for the chemists!

These similarities in structure mean that caffeine can bind to adenosine receptors and block their function. Adenosine on binding to its receptor generally inhibits physiological activity (it makes us feel more tired). Due to adenosine and its half brother caffeine having a similar molecular structure caffeine blocks adenosine receptors (mainly  A1 and A2 types of adenosine receptors) preventing their activation resulting in caffeine having a stimulant effect. Hence caffeine is an antagonist (It interferes with the binding and physiological affect of adenosine).

Still puzzled?

So if that’s perhaps gone over your head a little......

 Imagine you are putting together pieces of a puzzle. You are looking for a piece of the puzzle that joins to the piece you have in your hand (the adenosine receptor). You can see two similar looking pieces on the floor (an adenosine and caffeine molecule) that look like they'll both join onto the piece you are holding. You try piece A (the caffeine molecule) and find that it pretty much fits onto the piece you are holding (the receptor) but the fit is not perfect. Despite this you decide to continue putting the puzzle together. However you find that you cannot fit the rest of the puzzle together and complete it (receptor has not been activated so the physiological effect is inhibited-completing the puzzle) as the two pieces you first joined together are not the correct pieces to join to complete the puzzle (the physiological effect is inhibited).

You go back to the original piece you had in your hand (receptor) and this time you join piece B (the adenosine molecule) onto it and find that these two pieces fit perfectly together. This time you find you are easily able to complete the puzzle (the receptor is activated resulting in the full physiological effect-the puzzle is completed) as all the other pieces fit around the two you first joined together (the receptor and adenosine molecule).

Let’s put the puzzle together....

The binding of caffeine to adenosine receptors prevents adenosine from binding to the receptor causing a change in the cell's behaviour. Caffeine inhibits the effect of adenosine. This is how caffeine can make us feel more awake. As adenosine decreases neurotransmitter release, dilates blood vessel and inhibits lipolysis (break down of fats)-these effects make us feel more tired. Caffeine preventing this in turn makes us feel more awake.

Upcoming posts....

Caffeine and its effect on the physiological processes in our body- how this stimulant takes its effect on the nervous, gastrointestinal, respiratory systems and renal function.