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.

Would the Piano Stools please stand up?

Figure 1. A d6-piano stool complex for Iridium.

I have recently read an article from Chem. Commun. on the chemistry-biology interface [1]. It should be fascinating to both researchers in chemistry and biology alike, as the research is about the application of an artificial metalloenzyme in catalysis.

Modular in nature, the metalloenzyme consists of a host protein and the synthetic metal complex. In order to incorporate the metal catalyst to the protein host, a well-established way, for which the researchers have adopted, is the biotin-streptavidin technology.

The metal complex the researchers were using belonged to the class of d6-piano stool complex, and in this particular case, the metal was iridium (Figure 1). This type of piano-stool complexes seemed to be stable in biological environment, thus providing a benefit for the development of bio-catalysis. In order to make their catalyst more active in a biological environment, they have devised a ‘shielding strategy’, where they coated silicon nanoparticles as a protective layer on the metalloenzyme (Figure 2).

Figure 2. The design of the catalyst architecture.

The synthesis of the iridium complex involved the reaction of a protected TFA salt of the precursor di-amine with [Cp*IrCl₂]₂ in the presence of triethylamine at room temperature, resulting in the iridium catalyst with satisfactory yield (Figure 3).

Figure 3. Synthesis of the iridium complex.

The metalloenzyme, henceforth known as an ATHase, was covalently anchored onto the silicon nanoparticles (Figure 2). After self-assembly and the poly-condensation of silanes, a protective layer could be formed and embedded the ATHase inside the 'silica core'. The nanoparticles were visualized by scanning electron microscopy.

Figure 4. Catalysis of imine reduction by ATHase.

The ATHase was used to catalyze an imine reduction reaction (Figure 4). Depending on the specific mutant employed, it could give rise to a specific enantiomer as the product. In a nutshell, the conversion, enantioselectivity and turnover number of the silica-protected ATHase were impressive. What was notable was that, when the researchers compared the data of the catalysis of the metalloenzyme with or without silica-protection, the results were more or less comparable, signifying that the extra silica protection has not led to a negative impact to the catalysis. The silica-protected catalyst could be recycled and re-used, with almost the same enantiomeric excess in subsequent runs.

In terms of the context of the research aim, the authors would like to explore the applications of their ATHase in biological scenarios. Thus, they used the ATHase to carry out a reaction well known to anyone in biochemistry : the conversion of NAD⁺ to NADH. They have used absorbance spectroscopy (340 nm) – another obvious technique for biochemists – to monitor the conversion. They observed that the silica-protected ATHase had a somewhat better turnover number as compared to the non-protected version.

Another interesting aspect was that the researchers attempted to test their ATHase in vivo, which meant they did the reduction in a cellular ‘soup’. Using cell lysate of E. coli and other cellular media, satisfactory turnover numbers could be achieved, and the results were indeed better than the homogeneous metalloenzyme. They rationalized that, since the homogeneous metalloenzymes did not possess any protective layers, the catalytic activity was lost in the presence of cellular debris in the cellular ‘soup’. Thus, the silica ‘shell’ was able to protect the ATHase in the cellular media, while at the same time allowing the ATHase to carry out its mission. Even an imine reduction in urine (!!) could be achieved (TON= 4500)! 

The methodology has the added advantages that it could be conducted in physiological conditions – room temperature, pH 7, making it a stunning approach for bio-catalysis.

Impressive!

by Ed Law
20/8/2016

Reference:

1. Immobilization of an artificial imine reductase within silica nanoparticles improves its performance

Martina Hestericová, M. Rita Correro, Markus Lenz, Philippe F.-X. Corvini, Patrick Shahgaldian and Thomas R. Ward

Chem. Commun., 2016, asap

DOI: 10.1039/c6cc04604e