A new GPCR structure

The largest family of cell-surface receptors, the trimeric G-protein coupled receptors (GPCRs), are seven-pass transmembrane proteins that activate intracellular signaling pathways by switching conformations upon binding of an extracellular ligand. Although roughly half of all drugs function by affecting GPCRs, the only high-resolution GPCR crystal structure determined prior to this effort was that of rhodopsin. The primary problems in crystallizing GPCRs were thought to be the flexible, poorly-structured third intracellular loop (ICL3) and C-terminus. In this paper¹, the authors were able to crystallize the human β2-adrenergic receptor (β 2AR) by both truncating the C-terminus and removing ICL3, replacing it with T4 lysozyme (T4L), a small, well-structured protein that creates a polar surface similar to ICL3. This paper is important, in my view, because the protein engineering techniques used to crystallize this receptor could likely be used for other GPCRs, allowing expanded use of structure-based drug discovery for this family of proteins.

The first question the authors address is whether the engineered β 2AR (β 2AR-T4L) variant is similar enough to wild type β 2AR (WT β 2AR) for their structure to be meaningful. β 2AR-T4L has a two- to three-fold higher binding affinity for agonists and a partial agonist, and it appears to retain its capacity to switch conformations in response to ligand binding, based on the similarity of agonist-induced changes in fluorescence intensity of the fluorophore monobromobimane. They conclude that, due to the similarities of these properties to those known of GPCR constitutively active mutants, the fusion of T4L results in a constitutively active β 2AR phenotype. The overall structures of both unliganded β 2AR-T4L and β 2AR-T4L bound with the inverse agonist carazolol are also similar to the previously crystallized β 2AR-Fab complex. Replacement of ICL3 with T4L allowed the authors to crystallize β 2AR; however, ICL3 is important for both G protein specificity and activation. As predicted, β 2AR-T4L did not bind to the stimulatory G protein for adenylyl cyclase.

The authors are able to draw several insights into β 2AR function from their structure. First, the close packing of the helices on the cytoplasmic face of the receptor suggests that ligand-induced conformational changes occur through shifting of the side chains that interact between the helices. Second, they are able to map previously described mutations onto their structure: mutations leading to constitutive or impaired activation are located along the transmembrane helices, and none of these form part of the extracellular ligand-binding pocket. The structure shows that these residues are connected to each other and to the binding pocket by packing interactions, such that changes in the pocket due to ligand binding could be propagated through the transmembrane helices to the intracellular loops. Finally, β 2AR and rhodopsin share a set of conserved, loose-packed residues near a water molecule cluster that they propose is important in allowing conformational rearrangements due to low steric hindrance.

Left open by this work are the nature and dynamics of the multi-step GPCR activation. β 2AR, with a molecular weight of 38.9 kilodaltons, is a small enough protein that NMR relaxation dispersion experiments could be used to quantitatively probe the agonist-induced conformational changes. The process of β 2AR activation involves at least two distinct mechanisms²: (1) disruption of the 'ionic lock' interactions in the inactive structure between the cytoplasmic faces of helices II and III and helix VI, and (2) structural shifts around a conserved proline residue in helix VI (called a 'rotamer toggle switch'). Different agonists affect these molecular constraints in different ways: full agonists break the lock and trigger the switch, while partial agonists can alter one constraint but not the other. It should be possible to use β 2AR-T4L, which does not interact with the G protein, in the presence of excess agonist to limit the system to two-state exchange between the bound receptor-agonist complex and the complex after tripping both molecular switches. Likewise, β 2AR-T4L in the presence of excess partial agonist should create two-state conformational exchange between the receptor-ligand complex and the complex after tripping only one of the two switches. A potential complication is that, even at agonist saturation, β 2AR is known to sample several intermediate structures.

This structure also opens the door for the crystallization of other GPCRs by a similar method, and to further investigation of human β 2AR-T4L bound to different ligands. Another logical next step from this paper would be to attempt to co-crystallize the active state of the receptor in complex with an agonist ligand. The authors note, however, that efforts to crystallize β 2AR-T4L bound to catecholamine could be hindered by both the chemical instability of the catecholamine as well as the mutant receptor's lack of interaction with G protein, which results in relatively low agonist affinities.

References:

1. Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, Choi HJ, Yao XJ, Weis WI, Stevens RC, Kobilka BK. GPCR Engineering Yields High-Resolution Structural Insights into β 2-Adrenergic Receptor Function. Science 318: 1266-73 (2007).
2. Yao X, Parnot C, Deupi X, Ratnala VRP, Swaminath G, Farrens D, Kobilka B. Coupling ligand structure to specific conformational switches in the β 2-adrenoceptor. Nature Chemical Biology 2: 417-22 (2006).

Resveratrol!

Caloric restriction (CR) has been of interest for some time due to its apparent slowing of the aging process. In addition to conferring enhanced longevity, it is known to correlate with the alleviation of a number of biomarkers associated with aging, as well as lowering age-associated disease incidences. In spite of this, its mechanism remains poorly characterized. Resveratrol, a polyphenol with antioxidant and antitumorigenic activity, is intriguing because it seems to mimic many of the affects of CR, and has been shown to provide a variety of palliative and neuroprotective effects in several model organisms. In this work¹, the authors show that one function of resveratrol is to stimulate AMP kinase (AMPK) activity in neurons. This is a significant step towards characterizing resveratrol's mechanism, which, in turn, may help provide a better understanding of how CR works.

The authors show that in mouse Neuro2a neuroblastoma as well as mouse dorsal root ganglia (DRG) sensory and cortical neurons, resveratrol correlates with an increase in both phosphorylated AMP and a downstream target of activated AMPK, phosphorylated acetyl-CoA carboxylase (ACC). These increases are comparable to those generated by a well-characterized AMPK activator, AICAR. This AMPK activity is not stimulated indirectly by decreased cellular energy levels (measured by the relative AMP:ATP ratio). Next, they show that resveratrol-mediated AMPK activation halts Neuro2a cell proliferation and induces an increase in neurite outgrowth. This is again similar to the AICAR-mediated AMPK activation effects, and is consistent with AMPK's activity in other cell types. AMPK activation is required and sufficient for this resveratrol-induced neurite growth. AMPK activation is also correlated with mitochondrial biogenesis, as measured by increases in mRNA levels of three markers for mitochondrial biogenesis.

In Neuro2a cells, resveratrol’s activation of AMPK is unaffected by the presence of inhibitors for SIRT1 (associated with stress response and cell cycle regulation, and with polyphenol activities) or CaMKKβ (a kinase upstream of AMPK). This suggested that another activating kinase upstream of AMPK, LKB1, is likely to effect AMPK activation. To test this, DRG sensory and cortical neurons from mouse embryos containing loxP sites flanking the Lkb1 exon were infected with lentivirus expressing a Cre recombinase capable of conditionally knocking out Lkb1 from this engineered site. LKB1 removal reduced AMPK and ACC activation in both cell types. Interestingly, while introduction of a CaMKKβ inhibitor did not affect DRG sensory neurons, it inhibited AMPK and ACC activation in cortical neurons. Finally, the authors demonstrated that directly injecting mice with resveratrol within two hours led to increased levels of AMPK and ACC phosphorylation in the brain.

A major question raised by this work is the difference in resveratrol-mediated AMPK activation in Neuro2a and DRG sensory cells versus activation in cortical neurons. LKB1, which appears to regulate AMPK activation in both cell types, is part of the three-protein AMPK kinase complex, and is known to regulate AMPK response to decreased cellular energy levels (AMP buildup).² As shown in this work, resveratrol-induced AMPK activation is independent from LKB1’s response to cellular AMP levels. CaMKKβ appears to control AMPK response to Ca²⁺ levels in the cell, which raises the question of whether this is also the mechanism by which resveratrol induces AMPK activation in cortical neurons. This could be tested by examining CaMKKβ’s resveratrol-effected regulation of AMPK in varying concentrations of Ca²⁺.

Is there a physical interaction between resveratrol and either of upstream regulators of AMPK examined in this work? One way to test this would be through fluorescence resonance energy transfer (FRET). Attaching a fluorescent protein to LKB1 or CaMKKβ and a small fluorophore, such as Cy3 or Cy5, to resveratrol would result in emission at the small fluorophore peak if resveratrol is in close proximity to the regulatory kinase. Although the specificity of this technique, and the fact that it can be used in vivo, make it well-suited for this, a potential problem is that, due to resveratrol’s small size, attachment of a small fluorophore might alter its interactions significantly. Another possibility would be to label resveratrol, which has three hydroxyl groups, with a radioactive oxygen isotope, then use anti-LKB1 antibodies to immunoprecipitate the complex out of solution. If resveratrol is bound to the complex, Western blotting followed by film exposure of the blot would detect radioactive signal.

Another logical follow-up to this study would be to examine whether resveratrol and CR complement each other. Since resveratrol is predicted to function as a CR mimetic, the expectation is that introducing resveratrol into a system affected by CR would have no effect. There are several assays that could be done to gauge this by comparing CR mice with and without resveratrol to ad libitum fed mice with and without resveratrol. Since CR functions to increase life-span, the most straightforward measurement is mortality. Another, potentially more informative, assay is a comparison between AMPK and ACC activation levels between these four groups. In light of the differences in regulation of AMPK in different mouse neurons, it would make sense to do these assays for a variety of cell types, as it is possible that the CR and resveratrol pathways overlap for some cell types, but not others.

References:

1. Dasgupta B, Milbrandt J. Resveratrol stimulates AMP kinase activity in neurons. Proc. Natl. Acad. Sci. U.S.A. 104: 7217-22 (2007).
2. Shaw RJ, Lamia KA, Vasquez D, Koo SH, Bardeesy N, DePinho RA, Montminy M, Cantley LC. The Kinase LKB1 Mediates Glucose Homeostasis in Liver and Therapeutic Effects of Metformin. Science 310: 1642-46 (2005).

UC Bubble

I live in a bubble in Mission Bay, San Francisco.
Except it's not really San Francisco.
It's not even really Mission Bay.
It's just a bubble.

Fun with NAD

I stumbled on this interesting review article a short while ago: Hipkiss, AR. Energy metabolism, altered proteins, sirtuins and ageing: converging mechanisms? http://www.springerlink.com/content/j2332796374m3178/

I thought it led to some interesting predictions -- for example, what happens if you ramp up NAD levels? I've never worked with C. elegans but it strikes me that this might be an easy experiment to do with them, and you'd get results pretty quickly. According to this paper, you're getting this great benefit from increased NAD levels because it's used as a cofactor by an enzyme that converts glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate, which is important because G3P (and its precursor, dihydroxyacetone phosphate) can spontaneously decompose into the toxic glycating agent methylglyoxal. So what happens if you add more NAD into a system? Or, for that matter, increase levels and/or activity of the relevant enzyme (GAPDH)?

Untangling the interactions between telomeres, oxidative stress, and aging

In vitro human cells have a limited replicative capacity before they enter a non-dividing state, senescence. Senescence is inducible by the presence of artificially shortened telomeres. A number of immortalized cell lines contain the enzyme telomerase, which functions to add telomeric repeats to the distal tip of the telomere as they are lost. Introduction of telomerase into normal human cells in vitro extends the cells' replicative capacity indefinitely¹. Since telomere lengths are, on average, shorter in older humans than younger humans, this raises the question of whether telomere length plays a causal role in the aging process. There are, however, a number of facts that argue against this. Mice, for example, have very long telomeres that do not shorten with age, but they age rapidly compared to humans.

The reactive oxygen species (ROS) aging hypothesis states that cumulative oxidative damage causes aging. The results of experiments attempting to test this hypothesis have been mixed. Overexpression of two key antioxidative enzymes, superoxide dismutase (SOD) and catalase, in Drosophila melanogaster results in increased longevity; however, this effect only seems to exist in strains that are unusually short-lived². In Drosophila strains that naturally have a relatively long life-span, there is no increase in life-span due to increased antioxidant enzyme activity³. In Caenorhabditis elegans, antioxidant enzyme mimetics increase the life-span of the organism by an average of 44% in wild-type organisms⁴. In mice, a decrease in antioxidant enzyme activity is linked to an apparent increase in the rate of aging⁵.

When exposed to hydrogen peroxide, single-strand breaks accumulate in unusually high numbers on telomeres in cultured human cells due to a specific lack of function of normal DNA repair mechanisms on the telomeres. This translates directly into accelerated telomere attrition, which is ameliorated by the introduction of an ROS scavenger into the cells⁶. Therefore, telomere attrition in human cells is affected by oxidative stress. This creates a link between the telomere and ROS hypotheses of aging. The age-dependent telomeric length difference may be caused, at least in part, by accumulated ROS damage.

In C. elegans, it has been shown that telomere length and aging are independent of each other⁷. This then raises the question of what the general relationship is between ROS levels and telomere attrition. Does an increase in ROS level correlate with telomeric attrition rate in other organisms, and, if so, is that process coupled to aging, or is it an unrelated effect?

Aim 1: To measure telomeric DNA single-strand breaks and oxidative stress as a function of age in human fibroblasts in vitro.

Hypothesis: Telomeric single-strand breaks may show an ROS-mediated increase with age, due to the correlation previously observed between these two factors.

Experimental Approach: I propose to measure telomeric DNA single-strand breaks and ROS levels in a wide age range of human fibroblasts, the same cell type used by Von Zglinicki et al⁶. Assay of ROS levels will be done by measuring levels of superoxide anion, hydrogen peroxide, and hydroxyl radical via a chemiluminescence assay, as described by Aam and Fonnum⁸: the oxidation of luminol by ROS releases photons that can be measured with a luminometer. This assay is performed on live cell cultures and is non-toxic to the cells. A similar assay will be used to measure the levels of SOD. Oxidation of the purine base xanthine to uric acid by the enzyme xanthine oxidase generates superoxide anions. SOD functions to catalyze the breakdown of superoxide anion, so it is possible to gauge SOD activity by measuring the decrease in intensity of light from the reaction of these superoxide anions with luminol.

Telomere lengths will be measured by isolating and restricting genomic DNA from the fibroblasts and running the DNA out on an agarose gel. This will be followed by hybridization of Southern-blotted genomic DNA to a radiolabeled telomeric repeat sequence probe (5'-TTAGGG-3'). Telomeric single-strand breaks will be quantified by a nuclease protection assay, measuring the relative sensitivities to S1 nuclease, an endonuclease that cuts single-stranded nucleic acids, with a strong preference for DNA.

Outcomes: The results obtained here will provide a clear picture of how ROS levels affect telomere length with age in human fibroblasts. If there is a correlation between these measurements, this will indicate that there is a true link between the telomere and ROS aging hypotheses. This data will also present a starting point for unraveling the mechanism for the age-dependence of telomere length in human cells. If no correlation exists, this data will serve as another nail in the coffin for the already beleaguered telomere aging hypothesis.

Aim 2: Compare telomeric ROS and antioxidant enzyme activity levels to telomeric attrition in C. elegans as a function of age.

Hypothesis: Oxidative stress and telomeric attrition may not be coupled in C. elegans, as they are in in vitro human cells, so that there would not be a correlation between ROS/antioxidant enzyme measurements and levels of telomeric single-strand breaks.

Experimental Approach: The approach here will be essentially to replicate the experiment described in (6) as well as that in aim 1 for C. elegans. We will gauge telomeric DNA single-strand breaks and attrition rate for worms exposed to paraquat, an ROS generator, over the life-span of the worms. Assay of ROS levels will be done using the Amplex Red hydrogen/hydrogen peroxide assay, as described by Chavez et al⁹. Telomere length and single-strand break data will be gauged in the same way as for human cells in aim 1.

Outcomes: This data will give a clearer indication of what the interrelations between telomere attrition, oxidative stress, and aging are, and whether they are similar to these processes in human cells. Since telomere length is not age-dependent in C. elegans, if these results show a similar pattern as those for human cells, this would be a piece of evidence that the age-dependence of human telomeres is not a causal factor in aging. If the pattern is different, this indicates that aging in humans and in C. elegans follow different mechanisms in this respect, and that the decoupling of telomere length and age in C. elegans cannot necessarily be extrapolated to humans.

References:

1. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE. 1998. Science 279:349-52.
2. Orr WC, Mockett RJ, Benes JJ, Sohal RS. 2003. J Biol Chem. 278:26418-22.
3. Mockett RJ, Bayne AC, Kwong LK, Orr WC, Sohal RS. 2003. Free Radic Biol Med. 34:207-17.
4. Melov S, Ravenscroft J, Malik S, Gill MS, Walker DW, Clayton PE, Wallace DC, Malfroy B, Doctrow SR, Lithgow GJ. 2000. Science 289:1567-9.
5. Boldyrev AA, Yuneva MO, Sorokina EV, Kramarenko GG, Fedorova TN, Konovalova GG, Lankin VZ. 2001. Biochemistry (Mosc.) 66:1157-63.
6. Von Zglinicki T, Pilger P, Sitte N. 2000. Free Radic Biol Med. 28:64-74.
7. Raices M, Maruyama H, Dillin A, Karlseder J. 2005. PLoS Genetics 1:e30.
8. Aam BB, Fonnum F. 2007. Toxicology 230:207-18.
9. Chavez V, Mohri-Shiomi A, Maadani A, Vega LA, Garsin DA. 2007. Genetics 176:1567-77.

Yet another idea

How would increasing NAD levels in C. elegans affect their aging rate? Would this knock down their levels of MG?

Hmm...

Another idea

Most of my research as an undergraduate focused on telomeric length regulation. However, one of the limitations of the lab that I was working in at the time was that, due to financial constraints, we were not able to do protein work. One aspect that particularly interested me was better understanding the interactions between telomerase and the various proteins associated with the telomere.

The yeast K. lactis contains 12 telomeres that are composed of 10-20 repeats of a uniform 25 base pair sequence that is unique to the telomeres. The major telomeric length regulation pathway is known to be mediated by telomerase, which reverse transcribes telomeric repeats from its RNA template and concatenates them onto the end of the telomere. A major question regarding telomerase activity is what signals telomerase to replenish the telomeric tip. Telomere binding proteins have been shown to be crucial toward both recruiting telomerase to the end and inhibiting its access to the DNA.

One yeast telomeric protein clearly associated with the length-regulation mechanism is the Rap1 telomeric DNA-binding protein. Previous work indicates that telomere length in yeast is regulated through a pathway involving Rap1. Rap1 binds double-stranded sequence along the length of the telomere. Counting of Rap1 molecules at telomeres has been shown to be crucial to telomere length regulation. Each telomeric repeat of a variety of yeast species contains a Rap1 binding site. Telomere mutations disrupting Rap1 binding lead to greatly increased telomere length. The left portion of the K. lactis telomeric repeat is thought to have a negative regulatory function. Rif1 and Rif2 are accessory proteins that interact with the C-terminus of Rap1 and have also been shown to negatively regulate telomere length.

A variety of other proteins contribute to telomere maintenance in yeast. Cdc13 is a single-strand telomere binding protein that recruits or activates telomerase and also appears involved in negatively regulating telomerase. The MRX complex has been shown to be required for telomerase-mediated telomeric addition, probably by helping recruit telomerase to the telomeres. Two DNA damage response kinases, Tel1 and Mec1, also contribute to telomerase's ability to add new sequence onto telomeric ends. It has been shown that the Rap1-based repeat counting mechanism is at least partially dependent on Tel1 expression, but the nature of this epistatic dependence is not well characterized.

The goal here is to dissect the interaction between Tel1 and the Rap1/Rif telomerase regulation pathway. Some basic ideas I had on how I could do this:

- use FRET to observe the dynamics between the different components of this pathway
- attempt to crystallize components of this pathway, then try to dissect the pathway by computation (probably not practical, since there's quite a few relevant proteins, and Rap1 may have been crystallized, but I don't think any of the others have)
- generate multiple knockouts to figure out how many independent control points are involved in this pathway (e.g. cdc13/tel1 double deletion, what happens to the elongation phenotype?)
- use microarrays to determine which genes are being transcribed as the telomeres shorten
- use microarrays to determine how gene expressions differ between WT and mutants with runaway telomere elongation