Kubrick And Cholesterol

Figure 1

You may wonder what the greatest filmmaker in history has anything to do with that greasy little molecule – apparently the only common aspect is that both have something to do with Private Gomer Pyle (Figure 1). Here, I want to discuss an interesting concept about the cell membrane – ‘membrane fluidity’, and how cholesterol interacts with the membrane in different ways.

Membrane fluidity is defined as the viscosity of the lipid bilayer in either a cell membrane or a synthetic membrane. This property is important because it affects the lateral diffusion of the proteins molecules within the membrane bilayer, which is something based on the fluid mosaic model of membrane structure.

Figure 2

At physiological condition, the presence of cholesterol will decrease the membrane fluidity and hence increase the stability of the membrane bilayer (Figure 2). Because the structure of cholesterol is a rigid steroid system, it will interfere with the lateral movement of the fatty acid tails on the phospholipid molecules, and the membrane is therefore stabilized as a result. Nevertheless, a certain level of dynamic interactions is still present in the physiological range.

Yet, when the temperature of the system changes, the story ceases to be simple. This is a relevant point because the whole idea of membrane fluidity not only works in living things, but also for systems generated in vitro. That means the temperature is not necessarily in the physiological range, and it will be interesting to see what cholesterol will do to the membrane bilayers at different temperatures.

Cholesterol is actually a bidirectional regulator for membrane fluidity. At low or high temperature, its presence on a membrane bilayer will lead to opposing effects. 

At low temperature, molecular motions are restricted and the phospholipid molecules have limited motion, thus they are 'frozen out' and appear in a solid-like phase on the membrane (or 'gel-phase' as some may put it). In this case, the introduction of cholesterol will increase the fluidity of the membrane, making the phospholipid molecules more mobile as a consequence.

Figure 3

Thus, cholesterol is a bit like Barry Lyndon from the Stanley Kubrick film here (Figure 3). Barry, an underdog from the 18th century, was a party-crasher to the aristocratic class in his era. These rich guys only conformed to the rules and did not want to stand out from the crowd so as not to lose the prestigious status, as if in a form of stasis. The introduction of Barry to the upper class would certainly destabilize that, as seen in Figure 3.

On the other hand, when temperature is high, there are a lot of molecular motions, and the phospholipids are moving a lot on the membrane and the bilayer assumes a fluid-like phase. The introduction of cholesterol will decrease the membrane fluidity and stabilize the membrane bilayer.

Figure 4

Now, cholesterol looks more like Sergeant Hartman from Full Metal Jacket (Figure 4). No matter those new recruits are hippies, fanboys of Dennis Hopper, or still on a trip of LSD, Sgt. Hartman would certainly to whip up some discipline from these kids, or gut-punch any of them to put them in the right place, in a geometry very much like a ‘bilayer’.

For all membrane systems, there is a characteristic transition temperature, where the bilayer will change from a gel-like consistency to the fluid-like consistency (Figure 5). The value depends on the exact chemical structure of the types of phospholipid in question.

Figure 5

One thing for sure, though - no matter there is another war or not, Ann Margaret will probably not come anyway!

by Ed Law

6/10/2018

#CellMembrane #MembraneFluidity #Cholesterol

Hold Me Tight, Laser Jock

Don't you have, uh, ray guns?

-The Terminator (1984)

The Nobel Prize winners for Physics this year have all contributed significantly to the physics of Lasers - 'Light Amplification by Stimulated Emission of Radiation'. Laser is not merely related to a 'B coefficient' from Einstein, it has moved beyond the theoretical field to contribute in many areas. The wonderful aspect is that the winners' contributions to laser have demonstrated applications in molecular biology, chemistry and medical science alike. 

Professor Arthur Ashkin is awarded the Nobel Prize for the invention of optical tweezers. That is a laser-based approach for trapping and manipulating particles. Optical tweezers rely on the radiation pressure of light (which generates force) to do the job. While it may appear weird or may sound like something from an episode of Star Trek, radiation pressure exists in all electromagnetic radiation. While this type of pressure appears weak for a human being, it certainly is not so for a particle. Ashkin has discovered that a particle interacting with a laser beam will be pushed towards a center with the highest intensity, due to the action of radiation pressure. By using a lens to properly focus the laser light,  the particle is further drawn towards the point with the greatest laser intensity. In a sense, it looks as if the laser beam is 'holding' the particle at that point. Ashkin's light trap will eventually be called the 'optical tweezers' as we know nowadays.

It is through the use of laser that optical tweezers can be devised and they have been applied in fields like biophysics and chemical biology, where single molecule or particle-sized materials have to be 'held up' and investigated.

Figure 1. From Ref. [1].

A famous use of optical tweezers in biochemistry is the study of molecular motors of the cytoskeleton system (Figure 1). Kinesin, a motor protein which is involved in moving cargo along the microtubules, has served as a testament to the use of optical tweezers in molecular biology.  

The kinesin protein is first attached to a bead which is held up by the optical tweezers. When the kinesin starts moving along the microtubules, the bead is pulled along and it can be visualized and quantitatively established how far the molecular motor has gone. In a sense, the displacement of the bead is like its escape from the clutch of the optical tweezers. At the point when the kinesin has moved too far away, the bead will then be 'bounced back' into the focus point of the optical tweezer - like a spring restoring to its equilibrium.

Figure 2. From Ref. [2].

The other half of the Nobel Prize was awarded to the approach known as 'chirped pulse amplification', which has led to the generation of high intensity and ultra-short optical pulses. In a nutshell, it is a stress-and-compress process. First, an ultra short laser pulse is stretched in time, reducing its peak power. The pulse is linearly chirped through a dispersion, and it is amplified in a laser material, afterwards it is compressed in time to its original duration. The result is an significant increase in peak power.As a result, the laser pulses have enhanced intensity. 

The high intensity laser is important because whenever we want to deal with situations requiring good precision, the energy source must be very focused to minimize undesirable thermal energy around the target, leading to collateral damage. That is indeed why high intensity laser has been used in surgery, because the ultra short pulse can give very precise cuts on the area.  

Ultra short laser pulses can also be applied in spectroscopy. For example, the generation of charges in semiconductor devices can be monitored by spectroscopic approaches, yet there are challenges associated with that. Because the time scale of the photophysical processes are very short, therefore we need fast and accurate spectroscopic techniques – known as transient absorption spectroscopy, to deal with that. A well-established approach is known as the pump-probe spectroscopy, which often uses laser as an energy source. From the name, we can realize there are 2 beams that leads to the process. A pump beam is used to excite the (semiconductors) material to the excited energy state; and a probe beam is used to monitor the excited state. The advantage of pump-probe is that because the pump and probe beam can be fine-tuned, through the pulse length of the laser and the photon density of the probe beam, therefore it can monitor processes with a reasonable wide time-scale, leading to its versatility as a tool for photophysics.

All in all, it is great to see that the contributions from physics can also be nicely applied in chemistry and biology too!

by Ed Law

Reference:

1. http://www.iiserpune.ac.in/~cathale/lects/2011monsoon/bio322-student/014.pdf

2. Zeitoun et. al., X-ray Chirped Pulse Amplification: towards GW Soft X-ray Lasers

Appl. Sci. 2013, 3(3), 581-592.

Release the Immuno-Brake

The Nobel Prize in Physiology or Medicine has just been announced. Professor James Allison and Professor Tasuku Honjo have won the Nobel Prize for their revolutionary contributions to an important understanding in immunology and its potential application in cancer therapy. The topic has led to my fond memories of taking immunology lectures in my undergraduate years. Here, I will share with you the Nobel Laureates' contribution to the understanding of 'negative immune regulation' and its inhibition to unleash the powers of immune cells.
 
The concept of the immune system is extremely complex because it entails a lot of molecular and biochemical events, so I will just describe the key processes here. The immune system defends us from various sort of microbes, because it has the ability to distinguish 'self' (the host’s body) from 'non-self' (the microbes). One of the most important players in the immune system is the T-lymphocytes (‘T-cells’). The T-cell is further categorized into cytotoxic (CD8) T cells and helper (CD4) T cells.

The CD4 T-cell is a like a midfielder in a soccer game, because upon its activation, it will lead to the downstream processes for the other components of the immune system, such as cytotoxic (CD8) T cells, to deal with and hence eliminate the enemy. The CD4 T cells achieve this end by engaging in the process of antigen presentation, where they 'present' a fragment of the broken-down microbe to an antigen-presenting cell (such as dendritic cells, macrophages and B lymphocytes). The resulting molecular interaction leads to an activated response, which serves as a signal for the other immune cells to take further action. Furthermore, a co-stimulation is often required to lead to a full and total immune response, and this is achieved by some activator proteins.

So, what's the deal with a nice guy helping us around to deal with the party-crashers? Well, if the immune system goes way too far off the map, the result can be catastrophic. Through genetic, environmental, or pathological factors, the immune system may over-react and in turn attack the host's healthy cells because it is misled to act in that way. The result is a variety of autoimmune diseases, and many of these can have drastic consequences for the host.

The immune system has been evolved throughout humanity to deal with this potential pitfall. Immunologists have discovered a number of inhibitory proteins, which serve as ‘brakes’ to balance out the action of the activator proteins. It is through this mechanism that a regulation is possible for the immune system, and this tight control is a theme in many biological systems.

(Top) Under normal circumstances, the CTLA-4 and PD-1 proteins will inhibit the T-cell response and serve as brakes to the immune system in 2 different mechanisms. CTLA-4 competes the active site with the activatory proteins, while PD-1 inhibits the T-cell response by binding to an alternative site on another (in some cases, cancerous) cells. PD-1, which stands for Programmed Cell Death 1 protein, presents a more complex scenario because while it can prevent autoimmune disease by suppressing the immune system from overacting in a normal body, it will cause problems in a cancerous case because the immune system is suppressed by its action. (Bottom) Anti-CTLA-4 and Anti-PD-1 are used to block the action of the two brake proteins, releasing the immune system to take care of the cancerous cells.
[Source: https://www.nobelprize.org/prizes/medicine/2018/press-release/ ]

The two Nobel Prize laureates discovered respectively two proteins that inhibit the T-cell response with slightly different mechanisms.  The proteins are CTLA-4 and PD-1 respectively. These two proteins are inhibitory proteins that prevent full T-cell activation and hence they can regulate the activities of the T-cell and the immune systems in the normal circumstances. Known as a ‘negative immune response’, the two proteins serve as ‘brakes’ that prevent the immune system from pressing the ‘go’ button in the wrong scenario.

What do these discoveries have anything to do with cancer therapy, then? Over the past century, researches have tried to find different approaches for cancer therapy. What is rather ironic is that our guardian angel - namely our immune system - has not been successful in the contribution to cancer therapy. Some researchers have even gone as far as adopting Edward Jenner's logic of vaccination to provoke a 'cancerous immune response', yet their efforts prove futile.

Professors Allison and Honjo have respectively developed similar insights to harness the negative immune regulation. Because if there is a way to stop the action of the brake (CTLA-4 and PD-1), then the immune system can be unleashed in full force to do the job, especially in the face of serious disease such as cancer. The solution is to use a specific antibody that can bind to and inactivate the ‘brake’ proteins. Thus, antibodies, known as anti-CLTA-4 and anti-PD-1, have been developed, and both research groups have successfully demonstrated that the antibodies can inhibit the ‘brake’ proteins. The immune system is released from the grip of the ‘brake’ proteins and thus can turn NBK on the cancer cells. (In fact, there is a type of immune cells called Natural Killer cells!). The two teams have successfully proved their ideas in some animal models, and the discovery has led to the development of ‘immune checkpoint therapy’.

The beauty of the discovery lies in the fact that, through the understanding of an important regulatory mechanism, a novel approach to cancer therapy can be devised and that will benefit cancer patients in the long term. The experiments the researchers have carried out to lead to this discovery also serve as a testament to the caliber of modern molecular biology, an intensely fascinating field on its own right.

by Ed Law
1/10/2018

Dr. S, Rhodium and the Red Crystal

The new university semester for undergraduates around the world has just started recently. This time, I would like to share some of my fond memories about an inspiring chemistry teacher – Dr. S, and my indirect connection to the Nobel Prize-winning English chemist – Professor Geoffrey Wilkinson. While I will not explicitly state the identity of Dr. S, the smart readers will be able to find out somewhere in this article.

I have taken Chemistry as one of my A-Level subjects when I was studying in London about 10 years ago. One of my chemistry teachers, Dr. S, is someone who has since left a lasting impression on me, and he is probably one of the most admirable teachers I have ever met in school. He is knowledgeable, has a great British sense of humor, and is really committed to help and give advice to his students. I have had a lot of chances to interact with him during my A-Level studies. From all these interactions, I can feel his intense knowledge about the various aspects of chemistry, and furthermore, his wise insights about life. Often, he quite simply dictated the notes onto the whiteboard, without any reference to the textbooks, because, as he has said before, ‘I have taught this course for like 30 years.’ He loves his wife and family, and enjoys a decent glass of red wine. He has always demonstrated a genuine passion for his field. I still vividly remember the excitement he had when he was teaching my class about the various oxidation states of transition metal complexes, and the beautiful range of colors exhibited from those co-ordination compounds. His passion has infected me, too. He has motivated me to learn way beyond the boundary of exam syllabus, digging deep into the concepts of organic chemistry and co-ordination chemistry. These further studies have certainly benefited me in my future university work.

One great aspect regarding Dr. S is that, at the end of each lesson, he would always ask, ‘Does anyone have any questions or comments?’ To me, this is exactly the correct attitude to approach education. After all, the whole process of education should come from both directions, not merely about spoon-feeding some infants not knowing their places. The teacher can just learn as much as the students when the students are willing to provide feedback. The room to allow comments can provide a more liberal atmosphere for everyone, and can lead the students to pose appropriate questions and to generate discussions. This inspiration is something I have always borne in mind since then, and I still ask this to my students whenever I am serving as a teaching assistant in my department.

When I was studying chemistry in my undergraduate years, I had the opportunity to come across the wonderful work of the English chemist, Sir Geoffrey Wilkinson. By accident, I discovered an old research article of which Dr. S was co-authoring with Professor Wilkinson! Soon, I learnt that Dr. S was actually a PhD student from Professor Wilkinson’s research group in the early 1970s. Dr. S has told me on one occasion that he did his PhD in Imperial College, London, yet I have never drawn this connection myself. No wonder Dr. S has always been fascinated with transition metal chemistry! I also feel extremely fortunate that I can have such an indirect connection to Professor Wilkinson, someone I have always admired. Now, let me tell you a bit about Professor Wilkinson’s work, and Dr. S’s contribution to organometallic chemistry.

While Professor Wilkinson’s initial independent research career concentrated on nuclear chemistry, he soon decided to pursue his research in something that has fascinated him since his student years – the chemistry of transition metal complexes. Soon after, he worked in the US through the early 1950s. In Harvard, he and Woodward were instrumental in solving the structure of ferrocene, one of the most famous organometallic compounds in the chemistry world. In mid-1955, he was appointed to a chair professorship of Inorganic Chemistry in Imperial College, UK. He has been there ever since and has placed intense focus in the chemistry of transition metal compounds. He has worked a lot on the chemistry of ruthenium, rhodium and rhenium organometallic compounds, and the developments of many of these complexes have led to advances in the field of homogeneous catalysis. Other than ferrocene he has also led to the development of compounds like Wilkinson's catalyst, hexamethyltungsten and some elimination-stabilized alkyl complexes of transition metals.  His pioneering work in organometallic chemistry has led him to a Nobel Prize in 1973.  

Since last year, I have been working on rhodium-catalyzed hydroboration and hydroformylation reactions in my research, and have also been preparing analogous versions of the Wilkinson’s catalyst. Thus, I can totally appreciate the insightful achievement Professor Wilkinson has contributed to the field of homogeneous catalysis. Without his hard work, the development in organotransition metal chemistry would certainly less likely to be as vibrant as now. Indeed, I am proud of involving and contributing to this wonderful research field!

I am so fortunate to have met such an inspiring chemistry teacher, and more so, we can both stand on the shoulders of a giant, who encourages us to see further in the world of Chemistry. Thank you, Dr. S and Professor Wilkinson!

by Ed Law

4/10/2016

Reference:

1. A. Shortland and  G. Wilkinson

Hexamethyltungsten

J. Chem. Soc., Chem. Commun., 1972, 318.

DOI: 10.1039/C3972000318A

2. A. J. Shortland and G. Wilkinson

Preparation and properties of hexamethyltungsten

J. Chem. Soc., Dalton Trans., 1973, 872.

DOI: 10.1039/DT9730000872

Optoelectronic P DADdy

Figure 1. Synthesis of building block (1).

Speaking of Organic Electronics, I would like to share with you another recent article regarding some interesting organo-phosphorus conjugated compounds. [1]

In the paper, the researchers have developed synthetic routes towards novel phosphorus-containing π-conjugated compounds, by using an interesting phosphorus reagent known as (Tipp)P(SiMe₃)₂ (Figure 1). It was great to observe that the phosphorus compound (1) was not as air-sensitive as one might usually expect. Due to the presence of the two electron-withdrawing carbonyl groups, the yellow-colored compound (1) was stable enough to survive column chromatography.

Figure 2. Building up the conjugated pathway, as in (2).

On the other hand, the DAD (Donor-Acceptor-Donor) type compound (2) was synthesized by a Stille coupling at elevated temperature (Figure 2). It should be noted that the (mono or bis) tributylstannyl-thiophene is often a good candidate in these coupling reactions, especially when the thiophene units are need for organic electronics or functional materials. It is also interesting to note that the presence of the bulky TIPP group is essential for a successful coupling in this case.

X-ray crystallographic structures have been obtained for some of the compounds, and UV/vis spectra and cyclic voltammograms have also been acquired. When these compounds were compared with their corresponding nitrogen analogues, the LUMO energies of the organo-phosphorus compounds were found to be lower. The observations implied σ*-π* electronic couplings in these compounds. These phosphorus compounds were highly stable from TGA studies (likely due to the 2 carbonyl EWGs), which made them potential candidates for uses as organic materials.

Figure 3. Synthesis of the heavily conjugated organic molecule (3).

For the next stage, the compound (2) was used to synthesize the conjugated polymer (3) though sequential Stille coupling reactions (Figure 3). The researchers found that the optical bandgap of (3) was much narrower than its nitrogen counterpart. So, this polymer should be a great contender as a low bandgap conjugated polymer.

I like this paper because the novel organo-phosphorus compound looks interesting and useful to me. In the past, I have read about phospholes, and also some of the zirconium-based methods for their syntheses. A novel strategy has been used in this paper, and this approach is something I am not aware of. As a researcher working a lot with phosphines and phosphorus-based compounds, I find this article a rewarding one to read!

by Ed Law

27/9/2016

Reference:

1. Thieno[3,4-c]phosphole-4,6-dione: A Versatile Building Block for Phosphorus-Containing Functional π-Conjugated Systems 

Youhei Takeda, Kota Hatanaka, Takuya Nishida, and Satoshi Minakata

Chem. Eur. J., 2016

DOI: 10.1002/chem.201602392

Grassy Butterfly Dreams

Figure 1. Structure of the 'Butterfly' Compound.

I have always been interested in the field of Organic Electronics, and it is indeed a research field I would love to be involved in! To me, organic compounds are not merely about biological stuff. It is often the optical and electronic – optoelectronic – properties of the organic compounds that make them useful contenders as novel functional and electronic materials. Too often, it is through an understanding of the chemistry that we can fine-tune the chemical properties of the target compound in question, and lead to better functional materials for the future. I have recently read a paper about an organic material, and it is exactly bearing the above philosophy in mind. [1]

The compound has a potential as novel organic light-emitting diode (OLED) material (Figure 1). The compound can display the property of thermally activated delayed fluorescence (TADF), and for a good candidate in this field, the compound should  (a) have a small energy gap between the lowest singlet and triplet states; (b) a high intrinsic photoluminescence quantum yield (through orbital overlap) and (c) a relatively short delayed lifetime. From the data, the compound seems to do pretty great for all 3 requirements. Thus, this compound has a nice potential to serve as a green fluorescent OLED.

The shape and design of the green-colored compound are interesting. It resembles te shape of a butterfly, because the overall structure is a donor- π system-acceptor- π system-donor type (D-π-A-π-D). The donor group is phenoxazine (PXZ), while the acceptor group is a pyrimidine derivative.  

Figure 2. Buchwald-Hartwig Coupling approach towards the family of target compounds.

The synthesis of the compound was through a double Pd-catalyzed Buchwald-Hartwig amination of the phenoxazine nucelophile to a di-bromophenyl pyrimidine derivative (Figure 2). An X-ray crystal structure could also be obtained, and the molecular structure really resembled a butterfly! It is notable that the twisting angle between the PXZ and the phenyl ring is large, very much due to the steric hindrance provided from the PXZ component. This design is important because by the incorporation of a larger steric demand, it can lead to a spatial separation of the frontier molecular orbitals, lowering the singlet-triplet energy gap as a result (Criterion a).

Cyclic voltammograms have been obtained to probe the HOMO / LUMO energy levels of the butterfly compound. The absorption spectra of the compound provides nice insights into the electronic characters of the compound. An intense band with charge-transfer character signifies the transition from the electron-donating PXZ group to the electron-withdrawing pyrimidine unit. It is also noted that the absorption profiles overlap with that of a commonly used host material, CBP, meaning that in a doping system, effective energy transfer from CBP to this butterfly compound will be possible.

At the pyrimidine unit, there is a substituent at the 2-position, and this substituent can impact the property of the green-colored compound. It is found that the delayed fluorescence (DF) can be reduced by a bulkier 2-substituted group. This can suppress the non-radioactive decay and also other undesirable quenching processes due to triplet excitons, thus improving the performance at high luminance. 

After playing with the chemical aspects, the researchers then tested the novel compounds’ potential as TADFs in OLED devices. All the compounds lead to green light emission in the systems, possess good thermal stability, demonstrate very effective up-conversion (T1 > S1), achieve high intrinsic photoluminescence quantum yield, and perform well at high luminance conditions. The results are promising for the compounds involved.

Impressive work!

by Ed Law

26/9/2016

Reference:

1. Optimizing Optoelectronic Properties of Pyrimidine-Based TADF Emitters by Changing the Substituent for Organic Light-Emitting Diodes with External Quantum Efficiency Close to 25% and Slow Efficiency Roll-Off

Kailong Wu, Tao Zhang, Lisi Zhan, Cheng Zhong, Shaolong Gong, Nan Jiang, Zheng-Hong Lu, and Chuluo Yang, Chem. Eur. J. 2016, 22, 1 – 8.

DOI: 10.1002/chem.201601686

No RHO enSNAREs me

I have read the latest issue (August 2016) of Nature Reviews Molecular Cell Biology, and two of the articles on cell signaling are related to some of the topics I was highly fascinated in when I was doing biochemistry in my undergraduate years. The 2 review articles are about Rho family of GTPases and SNARE complexes. I recommend these articles to those studying in biochemistry or molecular cell biology in university.

The first article is about the regulation of Rho GTPases and the proteins that regulate this particular family of G proteins [1]. Rho proteins are important because it can mediate the changes in the structure of the actin cytoskeleton, when it is subject to a number of upstream signals originated from extercellular stimuli. When it is activated, the Rho protein can bind to a number of proteins that have action on actin, such as ROCK, N-WASP, MRCK, WAVE 2, to name a few examples. That will affect the G-Actin / F-Actin equilibrium, and affect the assembly or disassembly of the actin cytoskeleton. The downstream signaling can lead to effects such as cell migration, endosomal sorting, focal adhesion,  and the formation of cytoskeletal structures such as filopodia and lamellipodia.

I would illustrate with a quick example regarding actin action at the endosome (Figure 1). When Cdc42, a Rho GTPase, is activated, it will in turn activate 2 down stream proteins, N-WASP complex, and CIP4. N-WASP will in turn activate the next down stream protein, Arp2/3 complex. N-WASP, CIP4, Arp2/3 will associate with each other as a multi-protein complex around the internalizing endosome. F-actin will anchor itself at the protein complex, and it will draw the internalization of the endosome, facilitating the endocytosis. 

Figure 1. Example of Rho GTPase signalling.

The review points out that, not only the obvious GDP-GTP recycling can regulate the spatial-temporal action of the GTPase, there are other means that the G Protein can be regulated. The formation of specific protein complexes with the proteins associated with the action of GTPase (GEF, GAP, GDI), and post-translational modification (PTM), which the latter is a hot topic in chemical biology, are also possible means.   

I would like to draw your attention to the fact that, as stressed in the article, a phsophorylation on the Rho GTPase can have various effects on the signaling. It can inactivate the GTPase through a GTP-GDP recycling. It can mark the GTPase as a target for sequestration. It can also direct it towards the action of E3 ligase and lead it to protein degradation via the proteasome. Finally, it can lead to a subcellular localization, and in some case (such as RhoU), the GTPase will be directed towards the endosomal membrane, relevant for endocytic processes.

Figure 2. SNARE-mediated membrane fusion.

The second article is about the SNARE complex (Figure 2), which in most cases mediates intracellular membrane fusions [2]. The basic cycle is as follows. The 2 membranes, which have SNARE proteins on their respective surfaces as ‘contact points’, move towards each other. A docking event takes place and the SNARE proteins associate with each other to lock up the 2 membranes. The pulling action increases the membrane curvature, and ‘breaks’ up the inside by exposing the interior face of membrane. This leads to the contact between the distal sides of the 2 membranes, forming a fusion pore as a result. Finally, hen the curvature is relieved, the 2 membranes are fused together as a new membrane. After that, the SNARE complexes disassemble and can be recycled, via hydrolysis of ATP, to facilitate another round of membrane fusion. Though a basic picture is known, we have to be cautious that some of the detailed points are still controversial and demand further confirmation.

There are already a number of informative review articles regarding the SNARE complex [3], yet this one has a slightly different focus. It is found that, when the assembly of SNARE complex is carried out in vitro, it is far slower in rate than the effective process that takes place in vivo. This obviously suggests there are some missing components in the in vitro conditions that hinder that from being a good model for the real process. Thus, a number of techniques have been used to study and understand the action of the SNARE complex in vivo, and they are reviewed in the article. For example, X-ray crystallography has been used to study the complex formd from SNARE proteins and other accessory proteins (Sec1-Munc18 protein for example), shedding light on the mechanism. A particularly interesting X-ray structure is related to neuronal SNARE complex – its binding to Synaptotagmin 1, the synaptic protein that serves as a Ca²⁺ sensor and is involved in neuronal exocytosis and regulation of release of neurotransmitter. On the other hand, cryo-electron microscopy has also been employed to visualize the disassembly of SNARE complex by NSF and SNAP proteins. Of course, better attempts for the re-constitution of the cell systems in vitro has also led to a more realistic model for the in vivo process.

I have also found a further paper which links up the 2 concepts together, and it should be very useful for us to see how all these mechanisms (and other such as calcium and phosphoinositide signalling) working together in a cellular system [4].

Hope you find all these useful!

by Ed Law

23/8/2016

Reference :

1. Regulating Rho GTPases and their regulators, Richard G. Hodge and Anne J. Ridley,

Nature Reviews Molecular Cell Biology, 2016, 17, 496–510. doi:10.1038/nrm.2016.67

2. Chaperoning SNARE assembly and disassembly, Richard W. Baker and Frederick M. Hughson

Nature Reviews Molecular Cell Biology, 2016, 17, 465–479.

doi:10.1038/nrm.2016.65

3. For example, see: (a) SNAREs — engines for membrane fusion, Reinhard Jahn and Richard H. Scheller, Nature Reviews Molecular Cell Biology, 2006, 7, 631-643; (b) SNARE-mediated membrane fusion, Yu A. Chen and Richard H. Scheller, Nature Reviews Molecular Cell Biology, 2001, 2, 98-106.

4. The role of Rho GTPases and SNAREs in mediator release from granulocytes, Paige Lacy, Pharmacology & Therapeutics, 2005, 107, 358 – 376.