Abstract

Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that regulate the cellular levels of the second messenger molecules cAMP and cGMP by controlling their rates of degradation. In humans, there are 11 different PDE genes, and each gene produces several different isoforms and splice variants. The PDEs are a medically important class of proteins that regulate signaling across a range of tissues, and inhibition of these enzymes is clinically important in a wide array of human diseases. The human PDE3 gene produces two distinct, but related isoforms PDE3A and PDE3B. PDE3A is further subdivided into three isoforms with masses averaging at ~125 kDa. PDE3 play an important role in insulin signaling pathways, cardiovascular tissues, platelets, adipocytes, and oocyte maturation. In particular, PDE3A isoforms are prominently expressed in myocardium and vascular smooth muscle cells, hence, the inhibition of PDE3 activity is particularly useful in treatment of cardiac disease. The focus of this project is the PDE3 gene present in C. elegans. Although PDE3 activity has been widely studied from cell and tissue extracts from mammals, there is little data on purified protein for detailed biochemical analysis and characterization of the PDE3 gene and, based on the literature, it suggests that these are difficult proteins to express and purify. There is also very little structural data on the PDE family of enzymes. What exists is mostly on partial proteins, and none at all on the PDE3s.

The aims of this project were to clone and overexpress affinity-tagged recombinant proteins of isoforms of the C. elegans PDE3 gene to analyze their catalytic properties and attempt crystallization of the isoforms. The CEPDE3 gene is a homolog of the mammalian PDE3 family encoding two different CEPDE3 isoforms; CEPDE3-LF (long form) codes for a 63.5 kDa protein, and CEPDE3-SF (short form) codes for a 54.2 kDa protein. Sequence homology alignments of CEPDE3 with human PDE sequences indicate the mammalian and the nematode gene have ~ 47% homology. The advantages of this approach are that the C. elegans isoforms are smaller in mass, making them potentially easier to purify. Preliminary experimental data has suggested that the nematode CEPDE3 gene exhibits catalytic properties and inhibitor sensitivities that are characteristic of the mammalian PDE3 gene family. A crystal structure would enable the determination of structural elements of the PDE3 catalytic pocket which could contribute to the design of more effective small molecule modulators of PDE3 catalytic activity.

Initially, the CEPDE3-SF isoform was expressed as a His-tag protein from an existing clone. However, due to low protein expression, solubility, and enzyme activity, the use of a GST-tag was attempted since GST-tags are reported to improve solubility in some studies. Initial cloning using traditional PCR and restriction/ligation cloning was attempted to clone both isoforms into the plasmid pGEX-6P1 with little success. To overcome the problem, two alternative approaches to traditional cloning were attempted. The long form was amplified by PCR and was cloned by recombination using the TOPO vector system, prior to subcloning
into the expression vector pGEX-6P1. The short form was made synthetically and shipped as a codon optimized insert in a vector and also subcloned into the expression vector pGEX-6P1. Both isoforms were expressed as a GST-tag protein in E. coli and, despite the change of affinity tag, the solubility was still poor using standard expression conditions. The solubility of these proteins was a significant barrier to purification, and this was improved by the optimization of the growth conditions, IPTG concentration, and cell lysis. Optimal solubility was achieved by a novel expression protocol which included an incubation of cells at 4°C prior to expression. Both isoforms were then purified by optimizing conditions using the batch method of purification and using glutathione sepharose 4B. Cleavage of the GST-tag was achieved using PreScission Protease. The purified CEPDE3-LF and CEPDE3-SF after enzymatic cleavage from GST-tag correspond to the predicted size of 63.5 kDa and 54.2 kDa, respectively. Some initial biochemistry was performed on the proteins. CEPDE3-LF and SF lysate and purified protein were assayed for PDE3 enzyme activity by measuring breakdown of cAMP to adenosine. CEPDE3-SF purified protein showed an increase in enzyme activity (24 pmoles/min/mg) compared to crude lysate (5.6 pmoles/min/mg), which is a ~5-fold increase. CEPDE3-LF
showed an increase from 2.8 pmoles/min/mg in lysate to 37.5 pmoles/min/mg in purified protein; a ~ 7-fold increase in PDE3 enzyme activity. The CEPDE3-LF and SF isofoms expressed in Sf21 insect cells were assayed with a range of doses of the PDE3 specific inhibitor cilostamide to determine the capacity of enzyme inhibition. A 0.01 μM cilostamide
treatments showed a 10-20% inhibition of activity and 5 μM of cilostamide showed ~95% inhibition. Hence both isoforms were inhibited by PDE3 specific inhibitor. The CEPDE3 isoforms expressed in insect cells were also assayed for cAMP and cGMP hydrolysis. The CEPDE3-LF actively hydrolyzed cAMP substrate with a Vmax of 70.4 pmoles/min/mg, Km of 0.11
μM and Kcat of 0.26 seconds-1. In comparison, the CEPDE3-SF assay did not reach saturation under the same experimental conditions. Therefore, CEPDE3-SF was less efficient in cAMP hydrolysis. Meanwhile, CEPDE3-SF actively hydrolyzed cGMP substrate with a Vmax of 232 pmoles/min/mg, Km of 0.37 μM and Kcat of 0.84 seconds-1. However, CEPDE3-LF assay did not reach saturation under the same experimental conditions. While both CEPDE3 isoforms have dual specificity for cAMP and cGMP, in this study, the long isoform hydrolyzes cAMP more efficiently short isoform more efficiently hydrolyzes