Ilana Shoshani

Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA

The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein–protein and protein–DNA interactions within NER complexes. We have investigated an essential protein–protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1‐XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell‐free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.

2′,5′-Dideoxyadenosine 3′-Polyphosphates Are Potent Inhibitors of Adenylyl Cyclases

2′,5′-Dideoxyadenosine 3′-di- and triphosphates were tested as inhibitors of brain adenylyl cyclases. With an IC 40 nM, 2′,5′-dideoxy-3′-ATP is the most potent non-protein synthetic regulator of adenylyl cyclases thus far described. Neither 2′,5′-dideoxy-3′-ADP nor 2′,5′-dideoxy-3′-ATP inhibited activity by competition with substrate, and the linear noncompetitive inhibition observed was consistent with interaction via a distinct domain. The availability of this ligand will permit the development of a variety of probes that will be extremely useful in investigating adenylyl cyclase structure and the role(s) that this class of compound may play in physiologically regulating cell function.

Biophysical and Functional Analyses Suggest That Adenovirus E4-ORF3 Protein Requires Higher-order Multimerization to Function against Promyelocytic Leukemia Protein Nuclear Bodies

Background: The adenovirus E4-ORF3 protein disrupts PML nuclear bodies to inhibit antiviral activity. Results: The WT E4-ORF3 protein forms higher-order multimers, whereas a nonfunctional mutant forms a dimer. Conclusion: E4-ORF3 protein multimerization likely is required for the activity of this protein. Significance: These results provide new insight into the properties of the adenovirus E4-ORF3 protein and suggest that higher-order protein multimerization is essential for activity. The early region 4 open reading frame 3 protein (E4-ORF3; UniProt ID P04489) is the most highly conserved of all adenovirus-encoded gene products at the amino acid level. A conserved attribute of the E4-ORF3 proteins of different human adenoviruses is the ability to disrupt PML nuclear bodies from their normally punctate appearance into heterogeneous filamentous structures. This E4-ORF3 activity correlates with the inhibition of PML-mediated antiviral activity. The mechanism of E4-ORF3-mediated reorganization of PML nuclear bodies is unknown. Biophysical analysis of the purified WT E4-ORF3 protein revealed an ordered secondary/tertiary structure and the ability to form heterogeneous higher-order multimers in solution. Importantly, a nonfunctional E4-ORF3 mutant protein, L103A, forms a stable dimer with WT secondary structure content. Because the L103A mutant is incapable of PML reorganization, this result suggests that higher-order multimerization of E4-ORF3 may be required for the activity of the protein. In support of this hypothesis, we demonstrate that the E4-ORF3 L103A mutant protein acts as a dominant-negative effector when coexpressed with the WT E4-ORF3 in mammalian cells. It prevents WT E4-ORF3-mediated PML track formation presumably by binding to the WT protein and inhibiting the formation of higher-order multimers. In vitro protein binding studies support this conclusion as demonstrated by copurification of coexpressed WT and L103A proteins in Escherichia coli and coimmunoprecipitation of WT·L103A E4-ORF3 complexes in mammalian cells. These results provide new insight into the properties of the Ad E4-ORF3 protein and suggest that higher-order protein multimerization is essential for E4-ORF3 activity.

SYNTHESIS OF 2'-DEOXY- AND 2',5'-DIDEOXY-ADENOSINE-3'-DI- AND 3'-TRIPHOSPHATE

Abstract The synthesis of a number of adenine nucleotide-3′-polyphosphates has been devised, via a phosphotriester approach that combines the method of alkoxide activation with the use of 2,2,2-tribromoethyl phosphoromorpholinochloridate 2 as phosphorylating agent.

SYNTHESIS OF 2',5'-DIDEOXY-ADENOSINE-3'-MONOPHOSPHATE DERIVATIVES AS ALLOSTERIC INHIBITORS OF ADENYLYL CYCLASE

Abstract The synthesis of a fluorescent and a lipophilic conjugate of 2′,5′-dideoxy-3′-AMP, an allosteric inhibitor of adenylyl cyclase, has been devised.

Synthesis of Iodo-aryl-azido Adenosine Analogs as Affinity Ligands for Adenylyl Cyclase

Abstract Potential affinity probes for adenylyl cyclase were synthesized that take advantage of the enzymes sensitivity to “P”-site-mediated inhibition by 2′,5′-dideoxyadenosine analogs and its tolerance for large 3′-ribose substitutions. We report the synthesis of a series of 3′-substituted 2′,5′-dideoxyadenosine analogs. The syntheses involved the intermediate formation of symmetric anhydrides that were then coupled to 2′,5′-dideoxyadenosine by base-catalyzed esterification at the 3′-ribose position.