Enzymatic attributes of an l-isoaspartyl methyltransferase from Candida utilis and its role in cell survival

Abstract

Backgrounds: Spontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms.

Methods: PCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis.

Results: The 25.4 kDa native PCMT from C. utilis was found to have a K_m of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro.

General significance: Identification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.

Keywords: Deamidation; Enzyme catalysis; Enzyme purification; Isoaspartate; MALDI TOF; S-adenosyl l-methionine; Yeast.

Fig. 1

Mechanism of isoAsp repair by Protein Carboxyl Methyltransferase (PCMT). During prolonged storage at physiological pH, native proteins or peptides undergo either deamidation at l-asparaginyl residues or isomerisation and racemisation at l-aspartyl residues, resulting in the formation of abnormally linked l-isoaspapartates or d-aspartates. These damaged proteins or peptides exhibit reduced cellular functions. Protein l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) targets these l-isoAsp or d-aspartate residues to catalyze their selective methylation with the aid of methyl group donor S-adenosyl l-methionine (AdoMet). The methylation step leads to the formation of l-isoaspartyl or d-aspartyl methyl esters along with a molecule of S-adenosyl l-homocysteine (AdoHcy). A metastable succinimide intermediate is produced when the methyl esters lose a molecule of methanol (CH₃OH) spontaneously. The l-succinimide intermediate hydrolyzes to generate a mixture of l-isoaspartyl and l-aspartyl residues, or it spontaneously gets racemized into d-succinimide which upon hydrolysis results into a mixture of d-isoaspartyl and d-aspartyl residues. Each methylation cycle reduces the total l-isoaspartate (and d-aspartate) level in a protein or peptide population by 15–30% relative to the previous cycle, resulting in a net repair of ≥85% after 10 or more cycles. The active amino acid residues and the step leading from inactive abnormally linked residue to its active form are marked in green. The inactive residues and the steps leading to formation of an abnormally linked form of amino acid are marked in red.

Fig. 2

Nature of Isoaspartyl repair in C. utilis. (A) C. utilis cells were grown till early stationary phase. Cells were harvested and re-dissolved in fresh YPD media containing 1 mM oxidised adenosine periodate (AdOx), 30 µM MG-132 or 5 mM 3-methyladenine. Cells were incubated for 24 h. Cytosolic extracts were prepared and Isoaspartyl levels were detected as mentioned in methods Sections 2.3.1 and 2.3.2 respectively. Y-axis denotes pmoles of isoAsp per mg extract. (B) Cytosolic extracts of early stationary phase ΔpcmtC. utilispcmt cells were prepared and incubated at 37 °C for 72 h with various protease inhibitors. After incubation isoAsp content were detected for each set. X axis denotes the combination of protease inhibitors used along with Lysis Buffer (LB). Y axis denotes the isoAsp levels as pmoles of isoAsp per mg cytosolic extract. Data presented is from three independent experimentation sets. The horizontal bars indicate the average (Mean), and the error bars represent standard deviation (S.D.).

Fig. 3

Distribution of PCMT activity along the growth curve of C. utilis. X-axis denotes the time of incubation. Primary Y-axis denotes optical density of the culture at 660 nm. The resulting line graph is the growth curve of C. utilis. Secondary Y-axis denotes PCMT specific activity. Data represented is the mean of three independent experimentation sets and the error bars represent standard deviation (S.D.).

Fig. 4

Purification validation and molecular weight determination of purified CuPCMT. A. HPRPLC of purified PCMT in DeltaPak C4 column, Waters. The chromatogram denotes a single peak eluting at 21.5 min. X-axis denotes elution time in minutes and Y-axis denotes absorbance at 280 nm. The first peak is an artifact and was also observed in blank runs. B. HPSELC of the purified PCMT in Protein Pak 125 shows a single peak with retention time of 13.81 min corresponding to ~25 kDa according to the calibration curve of the column. C. SDS PAGE analysis was performed with 30 µg of purified CuPCMT protein as described in Section 2.3.8. Lane 1 shows low molecular weight standards from GE healthcare (Catalog no. 17-0446-01). The respective molecular weights f the standards are designated on the left of each band. 30 µg CuPCMT was loaded on to lane 2 which shows a single band with molecular weight of 25 kDa; D. MALDI TOF MS analysis of the whole purified protein exhibited a single spectrum indicating the true molecular weight of PCMT as 25.4 kDa.

Fig. 5

Temperature and pH dependence of purified CuPCMT. (A) Temperature stability of the native CuPCMT was determined by pre-incubating the enzyme proteins in different temperatures (10–90 °C) for 30 min and then measuring the enzyme activity at 30 °C. To obtain the temperature optima of the CuPCMT enzyme, the enzyme activity assay was conducted in different temperatures (10–90 °C). X-axis denotes the incubation temperatures and Y-axis denotes the CuPCMT specific activity in pmoles of methyl groups transferred per minute per mg protein. (B) pH stability of the native CuPCMT was determined by pre-incubating the enzyme proteins in different pH buffers (5.0–9.0) for 30 min and then measuring the enzyme activity at pH 6.8. To obtain the pH optima of the CuPCMT enzyme, the enzyme activity assay was conducted in different pH buffers (5.0–9.0). X-axis denotes the incubation pH and Y-axis denotes the CuPCMT specific activity in pmoles of methyl groups transferred per minute per mg protein. Data represented in the figure is the mean of three independent sets of experimentations and the standard deviation is denoted by error bars.

Fig. 6

Structural characteristics of the native CuPCMT. (A) Amino acid sequence of the CuPCMT was determined by MALDI TOF MS/MS analysis. The AdoMet binding sites within the sequence were determined by analyzing the sequence with online tool PSIPRED and are underlined in red; (B). Circular dichroism spectra of the purified PCMT. The X-axis represents the wavelength in nm and Y-axis represents molar ellipticity (θ). Deconvolution of the CD data by CDNN software gave percentages of the α-helix, β-sheet and random coil. The predicted and experimentally determined percentages are listed in Table 4; (C). To validate the amino acid sequence for structural characteristics, it was analyzed by CD search tool from NCBI. The output image denotes the conserved domains detected within the amino acid sequence; D. Phylogenetic comparisons between PCMT from Candida and eleven homologs from different representative organisms by Clustal Omega tool gave a Phylogenetic tree in the form of a rooted cladogram. The abbreviations used are: THEMA: Thermotoga maritima; HELPY: Helicobacter pylori; CANUT: Candida utilis; SCHPO: Schizosaccharomyces pombe; NEUCR: Neurospora crassa; TRIAE: Triticum aestivum; ARATH: Arabidopsis thaliana; CAEEL: Caenorhabditis elegans; DROME: Drosophila melanogaster; BOVIN: Bovine.

Fig. 7

In vitro repair efficiency of the native CuPCMT. Four candidate proteins were chosen based on their abundance in living organisms – two enzymes and two structural proteins. These proteins were aged in vitro and then subjected to a repair assay employing PCMT from C. utilis and AdoMet. IsoAsp levels of the control, aged and repaired proteins were also measured; (A) Enzyme reactivation assay for Hexokinase (Hx) demonstrates repair efficiency both in terms of enzyme activity as well as IsoAsp levels. The primary Y-axis denotes the enzyme activity in percentage where the activity of the native enzyme is designated as 100% and the secondary Y-axis represents isoAsp levels of the control, aged and repaired sets. (●) represent enzyme acivities and (─▲─) represents isoAsp contents. Data represented in the figure is the mean of three independent sets of experimentations and the standard deviation is denoted by error bars; (B) A similar representation for the enzyme reactivation assay of Glutamate dehydrogenase (GDH); (C) Repair of structural protein IgG was demonstrated in the form of CD spectra illustrating the changes in secondary structure upon aging and subsequent repair; (D) The isoAsp levels of control, aged and repaired IgG have been depicted along the Y-axis as pmoles of isoAsp/pmoles protein; (E) CD spectra comparison of the control, aged and repaired Cytochrome c (Cyt c) with Y-axis depicting the molar ellipticity (θ) and X-axis depicting the wavelength in nm; (F) The isoAsp levels of control, aged and repaired cytc have been depicted along the Y-axis as pmoles of isoAsp/pmoles protein. Data presented is from three independent experimentation sets. The horizontal bars indicate the average (Mean), and the error bars represent standard deviation (S.D.).

Fig. 8

In vivo regulation of CuPCMT in C. utilis. (A) C. utilis cells were grown up to early stationary phase and were incubated either with 0–2 mM AdOx or with 0–2 mM LiCl for 24 h. Post incubation cells were harvested, washed, lysed and checked for PCMT activity. Y-axis denotes PCMT activity in terms of pmoles of methyl groups transferred/min/ml enzyme. X-axis denotes both the concentration of AdOx and/or LiCl in mM. (B) ROS generation profile of cells treated by different concentrations of AdOx. The X-axis denotes the concentrations of AdOx used and the Y-axis denotes the percentage of cells showing H₂DCFDA fluorescence; (C) ROS generation profile of cells treated by different concentrations of LiCl. The X-axis denotes the concentrations of LiCl used and the Y-axis denotes the percentage of cells showing H₂DCFDA fluorescence; (D) Semi-quantitative RT-PCR analysis of CuPCMT transcript concentrations in cells incubated in different LiCl concentrations is exhibited in the top panel. β-actin transcript expression of the same sets of cells are designated in the lower panel; (E) Early stationary phase Candida cells were treated with 0.8 mM AdOx for 24 h followed by oxidative (), pH (), hyperosmotic () and heat () stress. Isoapartate levels of treated and untreated cells were worked out in terms of pmoles of isoAsp/pmole total cellular proteins. (F) Early stationary phase Candida cells were incubated in presence or absence of 1.2 mM LiCl for 24 h after which the cells were subjected to either oxidative (), pH (), hyperosmotic () and heat () stress. Intracellular isoAsp levels from control and treated sets were determined in terms of pmoles of isoAsp/pmole total cellular proteins. Data presented is from two independent experimentation sets. The horizontal bars indicate the average (Mean), and the error bars represent standard deviation (S.D.).

Fig. 9

Role of CuPCMT in cell survival under physiological stress. Control set represents early stationary phase C. utilis cells reconstituted in fresh YPD medium without AdOx or LiCl and not subjected to any stress condition. Untreated sets represent C. utilis cells reconstituted in media without AdOx or LiCl but subjected to the same stress condition as the treated sets. AdOx set represents C. utilis cells reconstituted in YPD medium containing 0.8 mM AdOx and subjected to stress condition. LiCl set represents C. utilis cells reconstituted in YPD medium containing 1 mM LiCl and subjected to stress conditions. Candida cells were subjected to oxidative stress by addition of 30 mM H₂O₂ for 200 min following 24 h incubation in either 0.8 mM AdOx or in 1 mM LiCl. Aliquots were drawn every 50 min. (A) Cell viability was assayed colorimetrically with MTT treatment and (B) cell death was monitored by flow cytometry analysis. Candida utilis cells were subjected to pH stress for with 0.1 mM Hydrochloric acid for 6 h following 24 h incubation in either 0.8 mM AdOx or 1 mM LiCl. Aliquots were drawn every 1 h. (C) Cell viability was assessed colorimetrically by MTT assay and (D) cell death by staining with Annexin V-FITC/PI followed by FACS analysis. C. utilis cells were subjected to hyperosmotic stress for 40 mins with 1.5 M NaCl following 24 h incubation in either 0.8 mM AdOx or in 1 mM LiCl. Aliquots were drawn at 10 min intervals from both treated and untreated sets and tested for (E) cell viability by MTT assay as well as (F) cell survival by flow cytometry after staining with Annexin V-FITC conjugate/PI. Candida utilis cells were subjected to heat stress at 42 °C for 40 min following 24 h incubation in either 0.8 mM AdOx or 1 mM LiCl. Aliquots were drawn every 10 min. (G) Cell viability was assessed colorimetrically by MTT assay and (H) cell death by staining with Annexin V-FITC/PI followed by FACS analysis.

Fig. 10

Identification of intracellular protein substrates of CuPCMT. Stationary phase cells were incubated for 24 h with or without 1 mM AdOx following which the cells were lysed and the lysate (100 µg) were subjected to two dimesional gel electrophoresis. The proteins were blotted onto a PVDF membrane and the methyl accepting proteins were identified following an on-blot (³H) methylation PCMT enzyme and AdoMet. Comparison between AdOx-and AdOx+extracts in A and B panels elucidate that inhibition of PCMT activity lead to increased accumulation of isoAsp moieties in cellular proteins. Methylated proteins are indicated by arrows. Corresponding spots in the gel were excised, trypsin digested and subjected to MALDI TOF MS/MS analysis. The possible downstream target proteins of PCMT from Candida are listed in Table 5.