The Structural Basis for the Phosphorylation-Induced Activation of Smad Proteins: a Dissertation

The Smad proteins transduce the signal of transforming growth factor-β (TGF-β) and related factors from the cell surface to the nucleus. Following C-terminal phosphorylation by a corresponding receptor kinase, the R-Smad proteins form heteromeric complexes with Smad4. These complexes translocate into the nucleus, bind specific transcriptional activators and DNA, ultimately modulating gene expression. Though studied through a variety of means, the stoichiometry of the R-Smad/Smad4 complex is unclear. We investigated the stoichiometry of the phosphorylation-induced R-Smad/Smad4 complex by using acidic amino acid substitutions to simulate phosphorylation. Size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry analysis revealed that the R-Smad/Smad4 complex is a heterotrimer consisting of two R-Smad subunits and one Smad4 subunit. In addition, a specific mechanism for phosphorylation-induced R-Smad/Smad4 complex formation was studied. Although it had been previously established that part of the mechanism through which phosphorylation induces Smad oligomerization is through relieving MH1-domain mediated autoinhibition of the MH2 (oligomerization) domain, it is also evident that phosphorylation serves to energetically drive Smad complex formation. Through mutational and size exclusion chromatography analysis, we established that phosphorylation induces oligomerization of the Smads by creating an electrostatic interaction between the phosphorylated C-terminal tail of one R-Smad subunit in a Smad trimer with a basic surface on an adjacent R-Smad or Smad4 subunit. The basic surface is defined largely by the L3 loop, a region that had previously been implicated in R-Smad interaction with the receptor kinase. Furthermore, the Smad MH2 domain shares a similar protein fold with the phosphoserine and phosphothreonine-binding FHA domains from proteins like Rad53 and Chk2. Taken together, these results suggest that the Smad MH2 domain may be a distinct phospho serine-binding domain, which utilizes a common basic surface to bind the receptor kinase and other Smads, and takes advantage of phosphorylation-induced allosteric changes dissociate from the receptor kinase and oligomerize with other Smads. Finally, the structural basis for the preferential formation of the R-Smad/Smad4 heterotrimeric complex over the R-Smad homotrimeric complex was explored through X-ray crystallography and isothermal titration calorimetry. Crystal structures of the Smad2/Smad4 and Smad3/Smad4 complexes revealed that specific residue differences in Smad4 compared to R-Smads resulted in highly favorable electrostatic interactions that explain the preference for the interaction with Smad4

A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties.

Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout

Differential scanning calorimetry stability comparison.

<p>Differential scanning calorimetry stability comparison.</p

PEGylation strategy and analysis.

<p>(A) Shows the three dimensional solvent accessible sites within the tetrameric crystal structure of <i>Arthrobacter globiformis</i> uricase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167935#pone.0167935.ref001" target="_blank">1</a>]. Each uricase monomer subunit of the tetrameric enzyme is shown in a different color, and residues selected for substitution with Cys (T11, N33, S142) are shown in yellow. (B) SDS-PAGE analysis of di-PEGylated and non-PEGylated uricase suggests that the di-PEGylated material runs at a much higher molecular weight relative to the non-PEGylated material. (C) A reverse-phase chromatography analysis of the di-PEGylated material suggests that 92.6% of the material is di-PEGylated, 4.4% mono-PEGylated, 0% unreacted and 2.7% over-PEGylated. (D) Demonstrates that the activity of di-PEGylated uricase is comparable to non-PEGylated uricase.</p

Expression of candidate uricase in <i>E</i>. <i>coli</i>. 8 uricase sequences were expressed in <i>E</i>. <i>coli</i>and evaluated for soluble (S) and insoluble (P) expression level.

<p>As shown in Fig 1, most uricases were present at high level in the insoluble (P) material. The pig-baboon chimera appears to express almost entirely in the insoluble (P) fraction (Lane 9). Cytosolic soluble (S) expression was considered favorable.</p

Mass Spec and SEC-LS Analysis of candidate uricases.

<p>Mass Spec and SEC-LS Analysis of candidate uricases.</p

In vivo rat PK and dog PK/PD assessment.

<p>(A) <i>In vivo</i> rat PK comparison of non-PEGylated (blue triangles), di-PEGylated uricase (black circles) and tri-PEGylated uricase (green inverted triangles) and Krystexxa^® (red squares). Rats were dosed IV at 5 mg/kg and samples were collected various time points and analyzed for residual uricase activity above background. Representative data from individual rats are shown. Both di-PEGylated and tri-PEGylated uricases have mono-phasic profiles. The half-life for the di-PEGylated uricase was ~22.8 hours and tri-PEGylated ~29.9 hours. Non-PEGylated uricase half-life in rats was 2–3 hours. Krystexxa^® (red squares) had an atypical initial elimination profile followed by a relatively linear PK profile. (B) <i>In vivo</i> canine PK/PD study of di-PEGylated uricase delivered via SC route of administration at 3 mg/kg (blue squares) or 10 mg/kg (red squares). Hashed lines are associated with the right axis and represent the % UA measured in the blood.</p

Enzymatic activity analysis in human serum.

<p>Substrate (UA) depletion assays were performed to assess uricase activity in 50% human serum. The rate (specific activity) of UA oxidation was calculated based on a linear decrease in absorbance. Solid (or dotted) lines represent Michaelis-Menten kinetic fits performed in Prism GraphPad. Di-PEGylated uricase activity was measured relative to Krystexxa^®.</p