Poly(ADP-ribose) polymerase in HeLa cells--a high resolution two-dimensional gel analysis

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Poly(ADP-ribosyl)ation is a crucial post-translational modification mediated by eukaryotic enzymes like PADPRP.
• This process involves the covalent attachment of poly(ADP-ribose) chains to target proteins, altering their function and localization.
• Understanding the heterogeneity of these modifications is essential for elucidating cellular processes such as DNA repair and gene regulation.

Purpose of the Study:

• To investigate the charge and size heterogeneity of auto-modified poly(ADP-ribose) polymerase (PADPRP) species.
• To identify and characterize other protein acceptors of poly(ADP-ribose) polymers within cellular lysates.
• To gain insights into the processing and modification of PADPRP in a cellular context.

Main Methods:

• High-resolution two-dimensional gel electrophoresis (2D-PAGE) of HeLa cellular lysates.
• Analysis using immunoblots with antibodies against PADPRP N-terminal peptides.
• In situ activity blot assays to detect auto-modifying potential and localization studies of ADP-ribosylated proteins.

Main Results:

• Extensive modification and processing of PADPRP were observed in HeLa cells.
• Multiple protein species immunoreactive to PADPRP antibodies were identified, including the native enzyme and a ~116 kDa protein.
• Several protein species demonstrated auto-modifying activity, and ADP-ribosylated proteins were localized within permeabilized cells.

Conclusions:

• PADPRP undergoes significant post-translational modification and processing, leading to charge and size heterogeneity.
• The study identified various protein acceptors of poly(ADP-ribose) polymers, highlighting the broad impact of this modification.
• These findings contribute to a deeper understanding of the complex regulatory mechanisms involving PADPRP and ADP-ribosylation.