ABSTRACT

1. Angel Li: ABSTRACT Protein synthesis is an essential part of the cell’s life cycle that requires the cooperation between proteins and ribonucleic acids in converting the cell’s genetic information into corresponding polypeptides. Translation initiation is an especially important regulatory step that determines whether synthesis proceeds, and can be potentially influenced by molecular inhibitors. It was hypothesized that the marine natural product pateamine A (PatA) interfered with this step by binding to initiation factors involved in ribosome recruitment. Current research revealed that PatA inhibited the proliferation of cancer cell lines by binding to the eukaryotic initiation factor 4A (eIF4A) to disrupt its normal function in translation initiation. Results indicate that the study of PatA may help to lend further insight into eukaryotic translation initiation as well as lead to the development of potential anti-cancer and anti-viral treatments.

2. BACKGROUND Translation is an important and complex process in which the cell synthesizes necessary proteins based on genetic information contained in the nucleus. It can be divided into three major stages: initiation, elongation, and termination, all of which require the proper functioning of ribonucleic acids as well as proteins (Campbell, 2002). The binding of ribosomal subunits to mRNA during initiation is an especially important rate limiting step and regulatory mechanism (Bordeleau, 2005). Elongation and polypeptide synthesis cannot occur prior to the completion of this initiation complex, making initiation the primary site for translation control (Low, 2005).

3. INITIATION FACTORS The formation of the initiation complex can occur through two distinct pathways. The first involves the recruitment of ribosomes to an internal ribosome entry site (IRES) on the mRNA. This process is cap-independent, meaning that binding does not require the presence of mRNA’s 5’ cap and oftentimes does not require any initiation factors. The second mechanism, however, is cap-dependent, and ribosomes are attached near the 5’ end of the mRNA template via ATP hydrolysis. This reaction is catalyzed by a class of translation initiation factors called eukaryotic initiation factors (eIFs) (Bordeleau, 2005). The translation initiation factor eIF4A is the prototypical member of the DEAD-box RNA helicase family (Bordeleau, 2005). RNA helicases from the DEAD-box family are present wherever RNA is found within the cell (Rocak, 2004). Existing in three different isoforms (eIFAI, eIFAII, eIFAIII), eIF4A collaborates with other eIF4s to act as an ATPase as well as a helicase in all types of RNA metabolism. These proteins utilize energy from ATP hydrolysis to carry out ribosome biogenesis, splicing, translation, as well as mRNA degradation (Bordeleau, 2005). As an enzyme, eIF4A activity is stimulated by eIF4G. As a helicase, eIF4A exists in free form and as a subunit of eIF4F complex, both of which are stimulated by eIF4B. However, eIF4G’s association with eIF4A is stable whereas eIF4B’s is not. Despite the in-depth understanding of eIF4A’s biochemical functions, its precise role in eukaryotic translation initiation is still not well-defined (Low, 2005).

4. PATEAMINE A In nature, many marine organisms protect themselves by producing toxic chemicals. Fortunately, scientists have been able to extract and utilize some of these chemicals in cellular research and drug development (Randall, 2001). Pateamine A (PatA) is derived from a New Zealand sea sponge and is well known for its immunosuppressive as well as anti-cancer effects (Downer, 2005). Researchers achieved total synthesis of the compound in 1998 and have continued to synthesize derivatives in hopes of finding a more stable compound with increased immunosuppressive activity. They also believed that a set of these derivatives may help to uncover the basis of PatA’s immunosuppressive and anti-cancer properties (Randall, 2001). There are already many chemical ligands known to interfere with prokaryotic translation, which have become useful antibiotics, as well as those that interfere with eukaryotic transcription, but the lack of small molecular inhibitors in eukaryotic translation symbolizes the potential importance of PatA in future clinical applications. It was hypothesized that PatA inhibits the growth of eukaryotic cells by binding to the translation initiation factor eIF4A (Downer, 2005).

5. RIBOSOME FORMATION Fig 1. Formation of the Initiation Complex in the Cap-Dependent Pathway

6. DISCUSSION • In that past year, 2 research groups working independently to study pateamine A came to the same conclusion: PatA interferes with eukaryotic translation initiation by binding to eIF4A. The findings were first published in the summer of 2005 by Jerry Pelletier, biochemistry professor at McGill University in Montreal, and his coworkers (Borman, 2005). Pelletier’s group pinpointed PatA after screening a library of marine extracts for new eukaryotic translation initiation inhibitors (Bordeleau, 2006). However, chemistry professor Daniel Romo of Texas A&M University, who published similar findings in December of 2005 in collaboration with pharmacology professor Jun O. Liu and coworkers of the Johns Hopkins School of Medicine, has been studying pateamine A for almost a decade. Romo and his lab completed the first total synthesis of PatA in 1998 and went on to work in conjunction with Liu’s lab to study its immunosuppressive properties, where researchers in Liu’s lab realized the compound’s effect on translation initiation (Borman, 2005). • Pateamine A is the first small molecule found to interfere with protein synthesis in eukaryotic cells, and both groups’ reports provide the initial insight into how it works to inhibit cell growth (Downer, 2005). PatA’s specificity is also novel in that it blocks initiation without disrupting any other step of translation. Both groups agreed that the binding of PatA to eIF4A results in a defect prior to the formation of the 48S complex at the initiation codon. However, Pelletier proposed that the defect occurs during the recruitment of 43S to mRNA while Liu-Romo believe that it occurs during initiation-codon scanning or recognition (Borman, 2005). Nevertheless, PatA functions by binding to eIF4A to increase its enzymatic activity and interfere with its ability to bind to its normal protein partners. In nature, eIF4A forms a stable complex with eIF4G while forming only temporary bonds with eIF4B. The binding of PatA causes eIF4A to form a stable complex with eIF4B instead, resulting in the cell’s production of ribosome-RNA clumps called stress granules. Cells usually produce stress granules to stop protein synthesis as a response to unfavorable conditions (Downer, 2005). The two groups are now studying the role of these clumps in inducing apoptosis, programmed cell death, and believe that the further testing of PatA could lead to its potential use in battling tumors and viral infections (Borman, 2005).

7. LIU-ROMO EXPERIMENTS • Fig 4. Identification of eIF4A as the Primary Target Protein of PatA • SDS-PAGE of B-PatA bound proteins from colon carcinoma RKO cell lysates • B-PatA pull-downs performed using streptavidin and indicated lysates • Confirmation of B-PatA target protein (eIF4A) in RKO and rabbit reticulocyte (RRL) cell lysates using immunoblotting

8. PATEAMINE A STRUCTURE Fig 2. Molecular Structure of PatA and Analogs

9. PELLETIER EXPERIMENTS • Fig 3. Specific Identification of eIF4A • eIF4A specifically bound to pateamine-Sepharose affinity matrix • Immunoblots of eIF4A isoforms using indicated antibodies

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