19 research outputs found
Fluorescence-Activated Cell Sorting of Human l‑asparaginase Mutant Libraries for Detecting Enzyme Variants with Enhanced Activity
Immunogenicity is
one of the most common complications occurring
during therapy making use of protein drugs of nonhuman origin. A notable
example of such a case is bacterial l-asparaginases (L-ASNases)
used for the treatment of acute lymphoblastic leukemia (ALL). The
replacement of the bacterial enzymes by human ones is thought to set
the basis for a major improvement of antileukemic therapy. Recently,
we solved the crystal structure of a human enzyme possessing L-ASNase
activity, designated hASNase-3. This enzyme is expressed as an inactive
precursor protein and post-translationally undergoes intramolecular
processing leading to the generation of two subunits which remain
noncovalently, yet tightly associated and constitute the catalytically
active form of the enzyme. We discovered that this intramolecular
processing can be drastically and selectively accelerated by the free
amino acid glycine. In the present study, we report on the molecular
engineering of hASNase-3 aiming at the improvement of its catalytic
properties. We created a fluorescence-activated cell sorting (FACS)-based
high-throughput screening system for the characterization of rationally
designed mutant libraries, capitalizing on the finding that free glycine
promotes autoproteolytic cleavage, which activates the mutant proteins
expressed in an <i>E. coli</i> strain devoid of aspartate
biosynthesis. Successive screening rounds led to the isolation of
catalytically improved variants showing up to 6-fold better catalytic
efficiency as compared to the wild-type enzyme. Our work establishes
a powerful strategy for further exploitation of the human asparaginase
sequence space to facilitate the identification of <i>in vitro</i>-evolved enzyme species that will lay the basis for improved ALL
therapy
Structures of Apo and Product-Bound Human l‑Asparaginase: Insights into the Mechanism of Autoproteolysis and Substrate Hydrolysis
Asparaginases catalyze the hydrolysis of the amino acid
asparagine
to aspartate and ammonia. Bacterial asparaginases are used in cancer
chemotherapy to deplete asparagine from the blood, because several
hematological malignancies depend on extracellular asparagine for
growth. To avoid the immune response against the bacterial enzymes,
it would be beneficial to replace them with human asparaginases. However,
unlike the bacterial asparaginases, the human enzymes have a millimolar <i>K</i><sub>m</sub> value for asparagine, making them inefficient
in depleting the amino acid from blood. To facilitate the development
of human variants suitable for therapeutic use, we determined the
structure of human l-asparaginase (hASNase3). This asparaginase
is an N-terminal nucleophile (Ntn) family member that requires autocleavage
between Gly167 and Thr168 to become catalytically competent. For most
Ntn hydrolases, this autoproteolytic activation occurs efficiently.
In contrast, hASNas3 is relatively stable in its uncleaved state,
and this allowed us to observe the structure of the enzyme prior to
cleavage. To determine the structure of the cleaved state, we exploited
our discovery that the free amino acid glycine promotes complete cleavage
of hASNase3. Both enzyme states were elucidated in the absence and
presence of the product aspartate. Together, these structures provide
insight into the conformational changes required for cleavage and
the precise enzyme–substrate interactions. The new understanding
of hASNase3 will serve to guide the design of variants that possess
a decreased <i>K</i><sub>m</sub> value for asparagine, making
the human enzyme a suitable replacement for the bacterial asparaginases
in cancer therapy
Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition
<div><p>Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the <i>ckβ</i> gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). <i>In vitro</i> phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.</p></div
Immunoblot detection of human and baker's yeast choline and ethanolamine kinases showing isoform specificity of CKα antiserum.
<p>Detection of purified hCKα1 (1), hCKα2 (2), hCKβ (3), Δ89N-hEK1 (4), hEK2α (5), Δ49N-hCKα2 (6), Δ84N-hCKα2 (7), yCK (8) and yEK (9) were performed with 10000-fold dilution of CKα antiserum. Each lane was loaded with 50 ng of purified protein. Lane M is ChemiBlot molecular weight marker.</p
Proteins and expression plasmids used in this study.
<p>Proteins and expression plasmids used in this study.</p
Effect of PKA phosphorylation and phosphorylation mimic mutation of CKβ on the sensitivity to HC-3 inhibition.
<p>The activities of unphosphorylated, <i>in vitro</i> phosphorylated, and S39D/S40D-mutant CKβ were measured by PK-LDH-dependent coupled-enzyme assays as described in the experimental procedures, with 4 mM choline as substrate and the indicated HC-3 inhibitor concentrations. Each bar represents the standard error of the mean (SEM) from three independent experiments.</p
Proteins and expression plasmids used in this study.
<p>pGEX-RB and pET-14b were used for expression as GST and 6x histidine fusion proteins, respectively.</p
Identification of Ser-39 and Ser-40 in hCKβ as the major sites of PKA phosphorylation.
<p>A) Schematic representation of potential sites of PKA phosphorylation in hCKβ as determined by mass spectrometry. <i>In vitro</i> phosphorylation of B) GST-hCKβ and GST-∆42NhCKβ recombinant fusion proteins, C) hCKβ, S39AhCKβ, S40AhCKβ, S42AhCKβ, S39A/S42AhCKβ, S40A/S42AhCKβ, and D) S39A/S40AhCKβ mutant proteins. The pictures are representative of at least two independent experiments. All CKβ mutant proteins were <i>in vitro</i> phosphorylated with 80 U PKA as described in the experimental procedures. Five micrograms of phosphorylated CKβ were loaded in each lane, and phosphorylation was detected with Pro-Q Diamond phosphoprotein stain. Total protein was stained with either Coomassie blue (B & D) or SYPRO<sup>®</sup> Ruby stain (C).</p
Effect of forskolin, IBMX, and H-89 treatment on the intracellular phosphorylation of GFP-CKβ.
<p>A) Phosphorylation state of the GFP-tagged wild type and S39A/S40ACKβ phosphorylation-negative mutant. B) Time-dependent effect of forskolin (fors) and IBMX treatment on the level of CKβ phosphorylation. C) Effect of H-89 treatment on the level of CKβ phosphorylation. In all experiments, the phosphorylation level of the immunoprecipitated CKβ was monitored by PhosphoPKAS antibody, and anti-GFP antibody detection was subsequently performed as the loading control. The intensities of respective bands were quantitated by Image J 1.42 software and plotted as the phosphorylation level relative to the control. Each bar represents the standard error of the mean (SEM) from two independent experiments. Statistical analysis was performed using one-way ANOVA and the Tukey HSD <i>post-hoc</i> test (*<i>p</i> < 0.05 vs control; **<i>p</i> < 0.01 vs control, #<i>p</i> < 0.05, significant between treatment group). M: Supersignal<sup>®</sup> molecular weight protein ladder.</p
Concurrent immunoblot detection of CKα1 and α2 isoforms in MCF-7 (lane 1) and HepG2 (lane 3) cell lysates.
<p>Only CKα1 was detected in the HCT-116 cell lysate (lane 2). 5 ng of each purified CKα1 and α2 were loaded as references (lane +). 50 µg of each cell lysate were loaded and detection was performed with 10000-fold dilution of CKα antiserum. Results are representative of triplicate experiments with similar results.</p