Fig 1.
Greatly simplified diagram of ceramide metabolism.
Key metabolites are shown in boxes; while enzymes are represented in italics. See Text for abbreviations.
Fig 2.
Structure of the proposed model of ceramide dynamics.
Left panel: Detailed pathway of ceramide biosynthesis and utilization. The pathway consists of three major subsystems: synthesis and utilization of DHC (left column), synthesis and utilization of PHC (right column), and fatty acid elongation and desaturation (center column). V1, V6, V11, V16, V21, V26, V30, V34, V38 and V42 represent fluxes catalyzed by ceramide synthase. V3, V8, V13, V18, V23, and V28, V32, V36, V40 and V44 represent fluxes catalyzed by dihydroceramidase and phytoceramidase, respectively. Reactions exiting the system to the left or right, namely V5, V10, V15, V20, V25, V29, V33, V37, V41 and V45, represent fluxes catalyzed by IPC synthase, while reactions entering the system from the left or right, V2, V7, V12, V17, V22, V27, V31, V35, V39 and V43, represent fluxes catalyzed by IPCase (Isc1). V4, V9, V14, V19, V24 represent DHC hydroxylase. The vertically shown reactions represent reactions catalyzed by other enzymes, such as remodelase (V46), fatty acid elongases (V47, V48, V50, V51, V52, and V53), and desaturase (V49). The boxes marked “IPC” summarily account for the complex sphingolipids IPC, MIPC, and M(IP)2C and their inter-conversions. This simplification seems reasonable as all three, IPC, MIPC and M(IP)2C, can serve as sources for the production of DHC and PHC. Right panel: The dynamic flux estimation is partly based on some fluxes whose magnitudes we took from our previous model [13]. The table summarizes these fluxes and indicates on the right how they constrain fluxes in our present model.
Fig 3.
Smoothed and interpolated concentrations of ceramide species.
The concentrations were reconstructed with the model, using inferred enzymatic activities; for raw data, see Material and Methods section. The X-axis represents the 30 minutes of heat stress, while the Y-axis represents the concentrations of pertinent ceramides. In each graph, red circles represent the smoothed and interpolated trends in experimental data, and the segmented blue lines represent the corresponding ceramide concentrations computed with the dynamic model using optimized enzymatic activities. The discontinuities are due to the optimization method, which was gleaned from multiple shooting methods. See Materials and Methods Section and Text for further details.
Fig 4.
Trends in ceramide synthase activities.
The upper panel shows ceramide synthase activities toward DHC, while the lower panel shows ceramide synthase activities toward PHC. The X-axis represents the 30 minutes of heat stress and the Y-axis represents fold changes in activities. In each plot, the blue and gray dots represent averaged and individual enzyme activities, respectively. The yellow and green shading marks similar enzyme activity patterns involved in DHC (top panel) and PHC (bottom panel) synthesis, respectively. It is unknown whether the concentrations of fatty acyl CoAs remain constant during the experiment. If they change dynamically, the shown trends refer to the combined changes in fatty acyl CoAs and ceramide synthase activities.
Fig 5.
Trends in ceramidase activities.
The upper and lower panels show activity trends of dihydroceramidase and phytoceramidase, respectively. The X-axis represents the 30 minutes of the heat stress experiment, while the Y-axis represents the fold changes in activities. In each plot, the blue and gray dots represent averaged and individual enzyme activities, respectively. The yellow and green shading marks similar enzyme activity patterns involved in DHC (top panel) and PHC (bottom panel) utilization, respectively.
Fig 6.
Trends in IPC synthase activities.
The upper and lower panels show IPC synthase activities from DHC to IPC and from PHC to IPC, respectively. The X-axis represents the 30 minutes of the heat stress experiment, and the Y-axis represents the fold change in activities. In each plot, the blue and gray dots represent averaged and individual enzyme activities, respectively. The yellow and green shading marks similar enzyme activity patterns involved in DHC (top panel) and PHC (bottom panel), respectively.
Fig 7.
The upper and lower panels show IPCase activities from IPC to DHC and from IPC to PHC respectively. The X-axis represents the 30 minutes of the heat stress experiment, and the Y-axis represents the fold change in activities. In each plot, the blue and gray dots represent averaged and individual enzyme activities, respectively. The yellow and green shading marks similar enzyme activity patterns involved in DHC (top panel) and PHC (bottom panel) side, respectively.
Fig 8.
Trends in DHC hydroxylase activities.
The X-axis represents the 30 minutes of the heat stress experiment and Y-axis represents fold-changes in activities. In each plot, the blue and gray dots represent averaged and individual enzyme activities, respectively. The yellow shading marks similar DHC hydroxylase activities from DHC toward PHC.
Fig 9.
Activities of fatty acid elongases, desaturase, and remodelase.
The X-axis represents the 30 minutes of the heat stress experiment and the Y-axis represents fold-changes in activities. In each plot, the blue and gray dots represent averaged and individual enzyme activities, respectively.
Fig 10.
Raw duplicate time series concentrations of ceramide species (connected blue symbols) and their means (red symbols).
The red curves show the smoothing spline interpolations of each dataset.
Fig 11.
Details of the procedures of flux estimation and enzyme activity estimation.
Fig 12.
Dynamic flux distributions of 10 reactions catalyzed by ceramide synthase.
Each grey dot represents one simulation. The blue line indicates the means, the black asterisks show the medians, and blue bars represent 20th and 80th percentiles (blue bars).
Fig 13.
Histograms of ceramide synthase fluxes from C16 DHS to C16 DHC (upper panel) and from C16 PHS to C16 PHC (lower panel).
Time 0 represents normal steady-state temperature conditions at the beginning of the heat stress experiment, which lasts for 30 minutes.
Fig 14.
Rough estimation of ceramide synthase activities.
Each grey dot represents one simulation. The blue line indicates the means, the black asterisks show the medians, and blue bars represent 20th and 80th percentiles (blue bars).