Glutamate Receptors and PD ?

Metabotropic Glutamate Receptors (mGluRs) have been shown to be involved in PD and are projected as important therapeutic targets with a neuroprotective approach for this aging disorder. The major focus for this class of GPCRs in its role in PD is through the non-cannonical non-dopaminergic pathway. Loss of Dopaminergic neurons in PD results in significant changes in other neurotransmitter systems (Glutamate and GABA) that play key roles in mediating mediating normal basal ganglia functions.

In a MPTP model of monkey, it has been demonstrated that antagonist MTEP (antagonist to mGluR5) shows anti-parkisonian effect and offers some degree of neuroprotection. This antagonist molecule is known to cross the blood-brain barrier and offer neuroprotection and this drug is cleared or flushed out very quickly. However, it is not clear what is the molecular mechanism of this mode of action. And the other question that crosses my mind is that how does this MTEP molecule really functions ? does it couple to the GPCR and induce the heterotrimeric G-protein signaling ? These are open questions and need to be explored more to figure out the mechanistic pathway.

GPCR crystallization : trick or science

The scientific community has been wondering about the know how for GPCR crystallization. It has took decades to arrive at the first structure of a GPCR: bovine rhodopsin. Though the structure was deciphered in 2000, the structure represented only an inactive form of the protein. Time took its toll and it was after another 8 years of hard work and dedication by brilliant scientists that the second structure of GPCR (the beta-2- adrenergic receptor) was published in 2008. The trick involved in introducing T4-lysozyme fused within the i3 loop of the receptor molecule to aid the stabilization of detergent solubilized receptor as well as the crystallization phenomenon. This structure also had the high affinity antagonist carazolol bound to it and thus defined the overall architecture of beta2AR and the structure of the ligand-binding pocket.

The Therapeutic Potential of LRRK2 and Alpha-synuclein in Parkinson's Disease

My review article with Dr. West just hit the Pubmed search today.....the full PDF is yet to be there....The article gives a broad overview on LRRK2 and alpha-synuclein with their potential role in Parkinson's disease and the therapeutic potential of these. The abstract of our article is as follows: Current treatments for Parkinson's disease fail to modify disease progression and the underlying pathogenic mechanisms remain elusive. The identification of specific targets responsible for disease will aid in the development of relevant model systems and the discovery of neuroprotective and neurorestorative therapies. Two promising protein candidates, alpha-synuclein and LRRK2, offer unique insight into the molecular basis of disease and the potential to intervene in pathogenesis. Although multiple lines of evidence support alpha-synuclein and LRRK2 as robust targets for therapy, the connection between protein function and neurodegeneration is unclear. Technology capable of mitigating alpha-synuclein and LRRK2 disease-associated function will ultimately be required before the true value of these proteins as therapeutic targets can be discerned.

GPCR activation and FRET

A recent mini-review article on G-protein and GPCR activation involving FRET study was nicely described by Vilardaga et al (Mol Endocrinol. 2009 Feb). It is a nice paper giving background on GPCR as well as the principles of FRET and describe the mechanisms of GPCr activation using this technology. The abstract of the paper states that : Many biochemical pathways are driven by G protein-coupled receptors (GPCRs), cell surface proteins that convert the binding of extracellular chemical, sensory, and mechanical stimuli into cellular signals. Their interaction with various ligands triggers receptor activation that typically couples to and activates heterotrimeric G proteins, which in turn control the propagation of secondary messenger molecules (e.g. cAMP) involved in critically important physiological processes (e.g. heart beat). Successful transfer of information from ligand binding events to intracellular signaling cascades involves a dynamic interplay between ligands, receptors, and G proteins. The development of FRET and BRET-based methods has now permitted the kinetic analysis of initial steps involved in GPCR-mediated signaling in live cells, and in systems as diverse as neurotransmitter and hormone signaling. The direct measurement of ligand efficacy at the level of the receptor by FRET is also now possible, and allows intrinsic efficacies of clinical drugs to be linked with the effect of receptor polymorphisms.

Finally Genentech and Roche shake hands

Directly quoted from news briefing as appeared at Yahoo Business:

Roche has agreed to acquire the 44 percent of shares in the U.S. firm it does not already own for $95 each, ending a long pursuit of the U.S. biotech group and its lucrative cancer drugs.

Even if clinical trial results with Genentech's cancer drug Avastin disappoint next month, the purchase of the world-leading U.S. biotech group makes sound financial sense.

Genentech is at the cutting edge of both biotechnology and cancer medicine -- exactly the place where all big drugmakers want to be as the flow of traditional drugs from research labs stalls and patents of today's blockbusters expire.

Big drugmakers have been seeking to diversify and reduce their reliance on slow-growing traditional prescription medicines, which face patent expiries and falling prices.

Roche's play for the remainder of Genentech will also yield synergies -- some $750-850 million a year before tax, according to Roche -- but the big prize is maximum exposure to the fastest-growing section of the global pharmaceuticals market.

Why GPCRs and membrane proteins migrate anomalously on SDS-PAGE ?

There has been a recent discussion about the migration pattern of membrane proteins and GPCRs in SDS-PAGE gels. From my experience on working with GPCRs for greater than 7 years, these proteins may a times have a tendency to run anomalously in SDS-PAGE gels.

Recent report explains the anomalous behavior being attributed to detergent. The abstract of the paper describes as: Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed "gel shifting," appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix ("hairpin") sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of -10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4-10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R(2) = 0.8), and with hairpin helicity (R(2) = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.

WIll the deal of Roche & Genentech see the light of the day ?

Following the encouragement of the buyout of Scheing-Plough by Merck, the drug giant Roche once again stirred up the discussion of acquiring Genentch. The two companies had earlier conversations which fell through due to a solid consensus on the pricing issue. Roche has now offered a price tag of $93 per share. If they still bump up the value by two dollar per share, the deal would be valued at $46.7 billion. Genentech authorities are looking for a value of more than $100 per share.

We will wait and watch how the deals goes and if at all it goes or not. The likelihood of it making through is rather high is what my feeling is.

Merck and Schering-Plough consolidate operations through acquisition

Drug maker giant Merck shedding off prior controversial ventures of Vioxx has announced the acquisition of rival drug maker Schering-Plough for a whooping $41 billion. The new deal will help consolidate the drug industry more on a solid footing. Perhaps, this news is a blow for the sales and marketing force as that sector might experience some lay-off resulting from this consolidation effort.

The new force will be in terms more effective in bringing discovery to a quicker platform with a renewed work force and bigger talent pool of brilliant scientists. The healthcare industry will probably benefit more out of this with more drugs coming out from a single big pharmaceutical giant.

This new deal follows on the heals of the Pfizer - Wyeth and probably opens the door for the Roche - Genentech to fall into place.

A new Orphan GPCR

The orphan g protein-coupled receptor 3 (GPR3) modulates amyloid-Beta Peptide generation in neurons.

Report convincingly showing the link between GPCR modulation and AD.

No ligand has yet been identified for this GPCR.

The abstract of the paper states: Deposition of the amyloid-beta peptide is a pathological hallmark of Alzheimer's disease. A high-throughput functional genomics screen identified G protein-coupled receptor 3 (GPR3), a constitutively active orphan G protein-coupled receptor, as a modulator of amyloid-beta production. Overexpression of GPR3 stimulated amyloid-beta production, whereas genetic ablation of GPR3 prevented accumulation of the amyloid-beta peptide in vitro and in an Alzheimer's disease mouse model. GPR3 expression led to increased formation and cell-surface localization of the mature gamma-secretase complex in the absence of an effect on Notch processing. GPR3 is highly expressed in areas of the normal human brain implicated in Alzheimer's disease and is elevated in the sporadic Alzheimer's disease brain. Thus, GPR3 represents a potential therapeutic target for the treatment of Alzheimer's disease.

Eukaryotic Membrane Proteins

Membrane proteins are extremely difficult to overexpress in an heterologous system. While E.Coli serves as a perfect host for expression, various difficulties render this system as an inconvenient tool of use.

Bacterial expression based on Escherichia coli has been one of the most widely studied because of its ease of availability, handling and scale up; and furthermore it is cheap to grow. However, this system has turned out not to be suitable for expression of mammalian transmembrane proteins due to the lack of post-translational modifications (e.g. glycosylation, fatty acid acylation, and phosphorylation) and the processing events required for correct folding of mammalian proteins. The
accumulation of expressed proteins in inclusion bodies (in a misfolded state) is yet
another drawback, as it is very difficult to recover functional protein. In addition,
insertion of the recombinantly expressed mammalian proteins into bacterial membranes
has caused toxicity in host cells.

YEASTS
Yeasts are unicellular eukaryotes which can easily be grown in large-scale cultures. They combine the advantages of short generation times (2 h) and growth in simple media. Easy handling and rapid genetic manipulation and the potential to perform eukaryotic post-translational modification renders yeast as one of the organisms of choice for over-expression. High levels of heterologous expression in yeast have been obtained with β2-adrenergic receptor, with the expression level being 115 pmol/mg of the membrane protein, for an O.F. 23. However, the level of expression of other GPCRs expressed in this system varies between 0.02 pmol/mg and 40 pmol/mg. Though yeast has been rather widely used for production of membrane proteins and GPCRs, there are four important issues. First, the yeast is surrounded by a tough cell wall which must be disrupted in order to extract the membranes and
intracellular proteins. In addition, protease-deficient strains should be used and protease inhibitors should be included during breakage of the cells. Second, the lipid composition of yeast membranes is different from that of mammalian cells. The low cholesterol content or even the presence of ergosterol results in altered binding properties of the expressed membrane proteins. Third, though the yeast system is capable of performing eukaryotic post-translational modification, the composition and quantity of N-glycans added by yeast are different from that of mammalian cells, and this can be problematic when specific oligosaccharide structures are essential for functionality. (Fourth and) finally, during large scale production, an appropriate ligand should be added to the growth medium and the pH should be maintained at the optimal value for the receptor in study.

A few other systems have been used for the expression of membrane proteins and GPCRs. Archaebacteria such as Halobacterium salinarium have been used to express two receptors successfully, the β2-adrenergic receptor and the yeast α-mating factor receptor. H.salinarium has the capacity to express large quantities of bacteriorhodopsin; and it was assumed that this system could express other transmembrane mammalian proteins. However, this was not the case and even chimeras of bacteriorhodopsin and mammalian transmembrane proteins resulted only in modest expression levels.

In addition to Baculovirus/insect cell, there are other insect systems.
For instance, the extracellular part of the luteinizing hormone (LH receptor) has been expressed in a non-functional form in a caterpillar expression system. In addition, there is a new system for expression of GPCRs in fly eyes. The Drosophila melonogaster metabotropic glutamate receptor (DmGluRA) was expressed by this novel strategy in good yields (170 μg/g fly heads) and was purified to homogeneity.

Insect Cells / BACULOVIRUS

The insect cell based baculoviral system provides an alternative approach for expression of GPCRs. The advantage of the baculovirus expression system is that it expresses the majority of membrane proteins and GPCRs in a functional form and at levels 10-100 fold higher than those observed in cells and tissues endogenously expressing them. In this system, the protein production results from infection of insect cells by recombinant viruses encoding the gene(s) of interest. The Baculovirus Autographica californica multiple nuclear polyhedrosis virus (AcMPPV) has been most widely used for the expression of membrane proteins in insect cells (usually using Spodoptera frugiperda, Sf9 cells). In most cases, the expression is driven by the polyhedrin or the p10 promoter, both of which are ‘very late’ promoters and are switched on only about 24 h post viral infection. The expression of the recombinant proteins under the control of very late promoters generally peaks 48-72 h post infection. The virus undergoes a lytic cycle and the cells die about 4-5 days post infection. The insect cells are easy to grow, can adapt to serum-free media and growth can be scaled up with ease. These cells can also perform post-translational modifications such as fatty acid acylation, phosphorylation and glycosylation. The recombinant proteins exhibit characteristics very similar to their native counterparts. In addition, co-expression of GPCRs and mammalian G-proteins is possible in this system allowing in vivo analysis of the specificity of interactions with G protein subtypes. A large number of membrane proteins and GPCRs have been successfully expressed using this system with levels as high as 10-
100 pmol/mg of membrane protein. The receptors which were recombinantly expressed
at such high levels include β-adrenergic, serotonin, muscarinic, dopamine, neurokinin
and chemokine receptors.
The expression levels sometimes depend on receptor subtype as well as the presence and position of a fusion tag. One drawback of this system is that it
often gives rise to heterogeneous proteins, arising out of poor glycosylation, which results in altered binding properties. Also, due to low cholesterol abundance in insect cells, alteration in GPCR binding has been observed with oxytocin receptor. Coexpression of various chaperones, peptidases, foldases and glycosylating enzymes has been shown to improve the secretion, processing and glycosylation of heterologous
proteins expressed in the baculovirus system.

GAPS and GEFs for LRRK2

As it is known that LRRK2 kinase activity is controlled by GTPase activity, there is a general query about the role of GEFs and GAPs in turning on/off of the GTPase activity. The partial crystal structure of LRRK2 - ROC domain indicates that the protein forms a dimer through generated by extensive domain-swapping. PD-associated mutants (R1441 and I1371) located at the interface of the two monomers provide exquisite interactions to stabilize the ROC dimer.

However, with the minimal quantity of LRRK2 expressed, it is not known which GEF's or GAPs play a role in the GDP/GTP exchange. There has not yet been and GDP-locked or GTP-locked structure / mutant of LRRK2 known. If someone comes up with either or both of them, a yeast-two hybrid approach can be used to find out the GAPs or GEFs.

LRRK2 story back again from my side

Keystone Symposium: Neurodegenerative Diseases: New Molecular Mechanisms

I was at this symposium very recently and got to hear very nice talks. The focus on the talk was mostly AD and HD and less emphasis on PD. The quality of presentations and the posters were very nice and of high scientific depth.

Several pharmaceutical companies were interested in LRRK2 poster of mine: looks like LRRK2 has been out there as a very attractive therapeutic drug target. And being a kinase, it attracts people very much to invest and investigate in order to arrive at a putative inhibitor.