Production and Crystallization of Full-Length Human AMP-Activated Protein Kinase (α1ß1γ1).

Determination of the crystal structure of AMP-activated protein kinase (AMPK) is fundamental to understanding its biological function and role in a number of diseases related to energy metabolism including type 2 diabetes, obesity, and cancer. We describe methods for the expression and purification of a human full-length active AMPK complex that is suitable for biochemical and structural analyses, followed by methods for its crystallization in complex with small molecule activators. Quality control of the purified protein by functional and biophysical analysis was an essential part of the process enabling the achievement of crystals of the full-length protein capable of being used for high-resolution structure determination by X-ray diffraction. X-ray structures have been determined of both phosphorylated and non-phosphorylated forms of full-length human AMPK α1ß1γ1.

Visualizing AMPK Drug Binding Sites Through Crystallization of Full-Length Phosphorylated α2ß1γ1 Heterotrimer.

Here, we describe the crystallization protocol for AMPK, including protein production and purification. AMPK can be readily crystallized in the presence of PEG to give diffracting crystals to a resolution of between 2.5 and 3.5 Å using synchrotron radiation. This method allows for visualization of drugs or small molecules that bind to the ADaM site, CBS sites, ATP binding site, and the newly identified C2 binding sites in the γ-subunit via co-crystallization with phosphorylated AMPK (pT172) α2ß1γ1 isoform or α2/1ß1γ1 chimera. Drugs with binding affinities above 500 nM fail to co-crystallize with AMPK using these parameters.

Biophysical Interactions of Direct AMPK Activators.

Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we describe some of the experimental methods that we have used to generate AMPK protein reagents and characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes were generated using standard molecular biology methods by expression either in insect cells via infection with three different viruses or more routinely in Escherichia coli with a tricistronic expression vector. Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry was used to probe protein conformational changes and potential binding sites of activators on AMPK. X-ray crystallographic studies were carried out on crystals of AMPK with bound ligands to reveal detailed molecular interactions formed by AMPK activators at near-atomic resolution. In order to gain insights into the mechanism of enzyme activation and to probe the effects of AMPK activators on kinetic parameters such as Michaelis-Menten constant (K m ) or maximal reaction velocity (V max), we performed classical enzyme kinetic studies using radioactive 33P-ATP-based filter assay. Equilibrium dissociation constants (K D ) and on and off rates of ligand binding were obtained by application of surface plasmon resonance (SPR) technique.

Transient Expression of AMPK Heterotrimer Complexes in Mammalian Cells.

Regulation of AMP-activated protein kinase (AMPK) signalling is complex and involves contributions from adenine nucleotides, co-/posttranslational modifications, and isoform composition of the AMPK heterotrimer. It is becoming apparent that AMPK activation/inhibition by synthetic drugs involves similar levels of complexity. Major advances in our understanding of these mechanisms have been gained from recombinant expression systems that provide sufficient quantities of highly purified material for structure/function studies. Here, we provide a detailed protocol for transient expression of affinity-tagged AMPK complexes in mammalian cells. We have found this system to be optimal as a source of enzyme possessing regulatory modifications found in vivo.

AMP-regulated protein kinase activity in the hearts of mice treated with low- or high-fat diet measured using novel LC-MS method.

AMP-regulated protein kinase (AMPK) is involved in regulation of energy-generating pathways in response to the metabolic needs in different organs including the heart. The activity of AMPK is mainly controlled by AMP concentration that in turn could be affected by nucleotide metabolic pathways. This study aimed to develop a procedure for measurement of AMPK activity together with nucleotide metabolic enzymes and its application for studies of mice treated with high-fat diet. The method developed was based on analysis of conversion of AMARA peptide to pAMARA by partially purified heart homogenate by liquid chromatography/mass spectrometry (LC/MS). Activities of the enzymes of nucleotide metabolism were evaluated by analysis of conversion of substrates into products by HPLC. The method was applied for analysis of hearts of mice fed 12 weeks with low- (LFD) or high-fat diet (HFD). The optimized method for AMPK activity analysis (measured in presence of AMP) revealed change of activity from 0.089 ± 0.035 pmol/min/mg protein in LFD to 0.024 ± 0.002 in HFD. This coincided with increase of adenosine deaminase (ADA) activity from 0.11 ± 0.02 to 0.19 ± 0.06 nmol/mg tissue/min and decrease of AMP-deaminase (AMPD) activity from 1.26 ± 0.35 to 0.56 ± 0.15 nmol/mg tissue/min for LFD and HFD, respectively. We have proven quality of our LC/MS method for analysis of AMPK activity. We observed decrease in AMPK activity in the heart of mice treated with high-fat diet. However, physiological consequences of this change could be modulated by decrease in AMPD activity.

Purification and characterization of truncated human AMPK alpha 2 beta 2 gamma 3 heterotrimer from baculovirus-infected insect cells.

AMP-activated protein kinase (AMPK) is considered an important target for treatment of type II diabetes and the metabolic syndrome. The muscle-specific isoform of the regulatory gamma-subunit, gamma 3, within the context of AMPK alpha 2 beta 2 gamma 3 complex, is involved in glucose and fat metabolism in skeletal muscle. In an effort to identify gamma 3-specific activators of AMPK, we have produced truncated human recombinant AMPK alpha 2 beta 2 gamma 3 (hu alpha 2 beta 2 gamma 3(trunc)) for biochemical characterization. Infection of insect cells with three baculoviral stocks encoding the individual subunits resulted in soluble expression of a stable hu alpha 2 beta 2 gamma 3(trunc) heterotrimeric complex. Co-expression of the three subunits was essential for solubility since the individual protein components, when expressed separately, were almost completely insoluble. The hu alpha 2 beta 2 gamma 3(trunc) heterotrimer was purified to apparent homogeneity from baculovirus-infected insect cells in a 1:1:1 stoichiometric complex. The hu alpha 2 beta 2 gamma 3(trunc) heterotrimer had significant circular dichroism signal that was lost as a function of temperature, implying that the purified protein was folded. The hu alpha 2 beta 2 gamma 3(trunc) complex was capable of binding AMP and ATP, although the heterotrimer showed preference for AMP, in particular, as seen by thermal denaturation circular dichroism analyses. Further experiments showed that the truncated complex bound ZMP (AICAR-monophosphate) albeit with much lower affinity than AMP. To investigate whether there were isoform-specific differences in the nucleotide binding affinities, a well-characterized truncated mammalian alpha 1 beta 2 gamma 1 (m alpha 1 beta 2 gamma 1(trunc)) equivalent of hu alpha 2 beta 2 gamma 3(trunc) was also purified. The gamma 1 and gamma 3 isoforms showed comparable nucleotide binding affinities and solution behavior properties.

Purification, crystallization and preliminary crystallographic analysis of protein MJ1225 from Methanocaldococcus jannaschii, a putative archaeal homologue of gamma-AMPK.

In mammals, AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (alpha) and two regulatory subunits (beta and gamma). The gamma subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. This work describes the purification and preliminary crystallographic analysis of protein MJ1225 from Methanocaldococcus jannaschii, an archaeal homologue of gamma-AMPK. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Diffraction data for MJ1225 were collected to 2.3 A resolution using synchrotron radiation. The crystals belonged to space group H32, with unit-cell parameters a = b = 108.95, c = 148.08 A, alpha = beta = 90.00, gamma = 120.00 degrees . Preliminary analysis of the X-ray data indicated that there was one molecule per asymmetric unit.

Tracking and quantification of 32P-labeled phosphopeptides in liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry.

Phosphoamino acid modifications on substrate proteins are critical components of protein kinase signaling pathways. Thus, diverse methodologies have been developed and applied to identify the sites of phosphorylated amino acids within proteins. Despite significant progress in the field, even the determination of phosphorylated residues in a given highly purified protein is not a matter of routine and can be difficult and time-consuming. Here we present a practicable approach that integrates into a liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry (LC-MALDI MS) workflow and allows localization and quantification of phosphorylated peptides on the MALDI target plate prior to MS analysis. Tryptic digests of radiolabeled proteins are fractionated by reversed-phase LC directly onto disposable MALDI target plates, followed by autoradiographic imaging. Visualization of the radiolabel enables focused analysis of selected spots, thereby accelerating the process of phosphorylation site mapping by decreasing the number of spectra to be acquired. Moreover, absolute quantification of the phosphorylated peptides is permitted by the use of appropriate standards. Finally, the manual sample handling is minimal, and consequently the risk of adsorptive sample loss is very low. Application of the procedure allowed the targeted identification of six novel autophosphorylation sites of AMP-activated protein kinase (AMPK) and displayed additional unknown phosphorylated peptide species not amenable to detection by MS. Furthermore, autoradiography revealed topologically inhomogeneous distribution of phosphorylated peptides within individual spots. However, accurate analysis of defined areas within single spots suggests that, rather than such quantitative differences, mainly the manner of matrix crystallization significantly affects ionization of phosphopeptides.

Are genuine changes in protein expression being overlooked? Reassessing Western blotting.

This study used purified calsequestrin 1 and AMP kinase (AMPK) proteins to demonstrate how Western blotting outcomes can be influenced when either the density of proteins detected lie within a nonproportional region of a standard curve or a standard curve is not taken into account for data analyses. It outlines the likelihood of true changes being overlooked through the simple mistake of using band density alone and/or through analyzing too much sample. To demonstrate this, extrapolation of a typical linear, although nonproportional, standard curve resulted in approximately fourfold error. The standard curve method was used to estimate the concentration of AMPK beta1 in rat extensor digitorum longus muscle as being of the order of 60 microM. The article suggests that adopting a more sensitive Western blotting protocol will improve the reliability of quantitative Western blotting outcomes.

Increased hepatic lipogenesis in insulin resistance and Type 2 diabetes is associated with AMPK signalling pathway up-regulation in Psammomys obesus.

AMPK (AMP-activated protein kinase) has been suggested to be a central player regulating FA (fatty acid) metabolism through its ability to regulate ACC (acetyl-CoA carboxylase) activity. Nevertheless, its involvement in insulin resistance- and TD2 (Type 2 diabetes)-associated dyslipidaemia remains enigmatic. In the present study, we employed the Psammomys obesus gerbil, a well-established model of insulin resistance and TD2, in order to appreciate the contribution of the AMPK/ACC pathway to the abnormal hepatic lipid synthesis and increased lipid accumulation in the liver. Our investigation provided evidence that the development of insulin resistance/diabetic state in P. obesus is accompanied by (i) body weight gain and hyperlipidaemia; (ii) elevations of hepatic ACC-Ser79 phosphorylation and ACC protein levels; (iii) a rise in the gene expression of cytosolic ACC1 concomitant with invariable mitochondrial ACC2; (iv) an increase in hepatic AMPKalpha-Thr172 phosphorylation and protein expression without any modification in the calculated ratio of phospho-AMPKalpha to total AMPKalpha; (v) a stimulation in ACC activity despite increased AMPKalpha phosphorylation and protein expression; and (vi) a trend of increase in mRNA levels of key lipogenic enzymes [SCD-1 (stearoyl-CoA desaturase-1), mGPAT (mitochondrial isoform of glycerol-3-phosphate acyltransferase) and FAS (FA synthase)] and transcription factors [SREBP-1 (sterol-regulatory-element-binding protein-1) and ChREBP (carbohydrate responsive element-binding protein)]. Altogether, our findings suggest that up-regulation of the AMPK pathway seems to be a natural response in order to reduce lipid metabolism abnormalities, thus supporting the role of AMPK as a promising target for the treatment of TD2-associated dyslipidaemia.