Search

Research shows that a hydrophobic surface patch on the C-terminal three-helix bundle motiff of the human GGA protein GAT domain directly binds rabaptin-5. Mutation in this hydrophobic patch reduces rabaptin-5 binding and a reverse mutation partially restores the binding. Furthermore, the binding of GAT domain to rabaptin-5 is not dependent on the ADP-ribosylation factor.

Research has been conducted on Golgi-localizing gamma-adapting (GGA1) GGA protein (GAT) domain which binds membrane-bound ARF proteins. The authors have investigated tha crystal structure of the human GGA1 GAT domain, and report that its structural characteristics support the hypothesis of the GAT domain multiple functional roles.

Poly(ADP-ribose) polymerase-1 (PARP-1) was recently identified as a platinum-DNA damage response protein. To investigate the properties of binding of PARP-1 to different platinum-DNA adducts in greater detail, biotinylated DNA probes containing a site-specific cisplatin 1,2-d(GpG) or 1,3-d(GpTpG) intrastrand cross-link or a cisplatin 5'-GC/5'-GC interstrand cross-link (ICL) were utilized in binding assays with cell-free extracts (CFEs) in vitro. The activated state of PARP-1 was generated by treatment of cells with a DNA-damaging agent or by addition of NAD(+) to CFEs. PARP-1 binds with a higher affinity to cisplatin-damaged DNA than to undamaged DNA, and the amount of protein that binds to the most common cisplatin-DNA cross-link, 1,2-d(GpG), is greater than the amount that binds to other types of cisplatin-DNA cross-links. Both DNA damage-activated PARP-1 and unactivated PARP-1 bind to cisplatin-damaged DNA, and both automodified PARP-1 and cleaved PARP-1 bind to cisplatin-DNA lesions. The role of poly(ADP-ribose) (pADPr) in mediating binding of PARP-1 to platinum damage was further investigated. The extent of binding of PARP-1 to the cisplatin 1,2-d(GpG) cross-link decreases upon automodification, and overactivated PARP-1 loses its affinity for the cross-link. Elimination of pADPr facilitates binding of PARP-1 to the cisplatin 1,2-d(GpG) cross-link. PARP-1 also binds to DNA damaged by other platinum compounds, including oxaliplatin and pyriplatin, indicating protein affinity for the damage in an adduct-specific manner rather than recognition of distorted DNA. Our results reveal the unique binding properties for binding of PARP-1 to platinum-DNA damage, providing insights into, and a better understanding of, the cellular response to platinum-based anticancer drugs.

The DNA-binding inorganic compound cisplatin is one of the most successful anticancer drugs. The detailed mechanism by which cells recognize and process cisplatin-DNA damage is of great interest. Although the family of proteins that bind cisplatin 1,2- and 1,3-intrastrand cross-links has been identified, much less is known about cellular protein interactions with cisplatin interstrand cross-links (ICLs). In order to address this question, a photoreactive analogue of cisplatin, PtBP(6), was used to construct a DNA duplex containing a site-specific platinum ICL. This DNA probe was characterized and used in photo-cross-linking experiments to separate and identify nuclear proteins that bind to the ICL by peptide mass fingerprint analysis. Several such proteins were discovered, including PARP-1, hMutSbeta, DNA ligase III, XRCC1, and PNK. The photo-cross-linking approach was independently validated by an electrophoretic mobility shift assay demonstrating hMutSbeta binding to a cisplatin ICL. Proteins that recognize the platinum ICL were also identified in cisplatin-resistant cells, cells halted at various phases of the cell cycle, and in different carcinoma cells. Nuclear proteins that bind to the platinum ICL differ from those binding to intrastrand cross-links, indicating different mechanisms for disruption of cellular functions.

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K(d), values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the K(d) decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.