RESUMO
Natural products are a valuable source of pharmaceuticals, providing a majority of the small-molecule drugs in use today. However, their production through organic synthesis or in heterologous hosts can be difficult and time-consuming. Therefore, to allow for easier screening and production of natural products, we demonstrated the use of a cell-free protein synthesis system to partially assemble natural products in vitro using S-Adenosyl Methionine (SAM)-dependent methyltransferase enzyme reactions. The tea caffeine synthase, TCS1, was utilized to synthesize caffeine within a cell-free protein synthesis system. Cell-free systems also provide the benefit of allowing the use of substrates that would normally be toxic in a cellular environment to synthesize novel products. However, TCS1 is unable to utilize a compound like S-adenosyl ethionine as a cofactor to create ethylated caffeine analogs. The automation and reduced metabolic engineering requirements of cell-free protein synthesis systems, in combination with other synthesis methods, may enable the more efficient generation of new compounds. Graphical Abstract.
RESUMO
Protein modification by SUMO helps orchestrate the elaborate events of meiosis to faithfully produce haploid gametes. To date, only a handful of meiotic SUMO targets have been identified. Here, we delineate a multidimensional SUMO-modified meiotic proteome in budding yeast, identifying 2747 conjugation sites in 775 targets, and defining their relative levels and dynamics. Modified sites cluster in disordered regions and only a minority match consensus motifs. Target identities and modification dynamics imply that SUMOylation regulates all levels of chromosome organization and each step of meiotic prophase I. Execution-point analysis confirms these inferences, revealing functions for SUMO in S-phase, the initiation of recombination, chromosome synapsis and crossing over. K15-linked SUMO chains become prominent as chromosomes synapse and recombine, consistent with roles in these processes. SUMO also modifies ubiquitin, forming hybrid oligomers with potential to modulate ubiquitin signaling. We conclude that SUMO plays diverse and unanticipated roles in regulating meiotic chromosome metabolism.
Most mammalian, yeast and other eukaryote cells have two sets of chromosomes, one from each parent, which contain all the cell's DNA. Sex cells like the sperm and egg however, have half the number of chromosomes and are formed by a specialized type of cell division known as meiosis. At the start of meiosis, each cell replicates its chromosomes so that it has twice the amount of DNA. The cell then undergoes two rounds of division to form sex cells which each contain only one set of chromosomes. Before the cell divides, the two duplicated sets of chromosomes pair up and swap sections of their DNA. This exchange allows each new sex cell to have a unique combination of DNA, resulting in offspring that are genetically distinct from their parents. This complex series of events is tightly regulated, in part, by a protein called the 'small ubiquitin-like modifier' (or SUMO for short), which attaches itself to other proteins and modifies their behavior. This process, known as SUMOylation, can affect a protein's stability, where it is located in the cell and how it interacts with other proteins. However, despite SUMO being known as a key regulator of meiosis, only a handful of its protein targets have been identified. To gain a better understanding of what SUMO does during meiosis, Bhagwat et al. set out to find which proteins are targeted by SUMO in budding yeast and to map the specific sites of modification. The experiments identified 2,747 different sites on 775 different proteins, suggesting that SUMO regulates all aspects of meiosis. Consistently, inactivating SUMOylation at different times revealed SUMO plays a role at every stage of meiosis, including the replication of DNA and the exchanges between chromosomes. In depth analysis of the targeted proteins also revealed that SUMOylation targets different groups of proteins at different stages of meiosis and interacts with other protein modifications, including the ubiquitin system which tags proteins for destruction. The data gathered by Bhagwat et al. provide a starting point for future research into precisely how SUMO proteins control meiosis in yeast and other organisms. In humans, errors in meiosis are the leading cause of pregnancy loss and congenital diseases. Most of the proteins identified as SUMO targets in budding yeast are also present in humans. So, this research could provide a platform for medical advances in the future. The next step is to study mammalian models, such as mice, to confirm that the regulation of meiosis by SUMO is the same in mammals as in yeast.
