This web page was produced as an assignment for Gen677 at UW-Madison Spring 2009
Future directions toward a better understanding of SIRT6
With my better understanding of SIRT6 and its biological role in the cells of organisms I feel there are some key areas of research that need to be addressed for SIRT6 role to be truly understood at both a molecular and physiological level. In this page I hope to accomplish that by suggesting some key experiments that I believe could bring about this better understanding of SIRT6.
Molecular Level
I feel there are some of the critical first questions to be asked about SIRT6 at the molecular level is 1) how is it controlled post-tanslationaly 2) what are other proteins it interacts with 3) what are other substrates that SIRT6 deacetylates.
To address my first question about how SIRT6 is controlled post translationaly would be to look at if and how SIRT6 is modified. Such as phosophorlation, sumolyation, and ubiqutinated, all or any of these could all SIRT6 to become more active or less or even for SIRT6 to be degraded. The first possible way I would look for SIRT6 to be modified would to be to just in vitro see if SIRT6 can be modified first. This could be done simply by incubating SIRT6 with a kinase or E1/E2 sumo enzymes and then simply run a western blot and probe for these possible modifications. But to see if SIRT6 is modified in vivio, would to be use cells that where serum starved (done to activate SIRT6) and normal cell and run a mass spec analysis looking for in vivio modification and there possible location. You could also use those same cells and run a simple western to see if SIRT6 is even modified. Once you did have a possible site of modification you could then due site directed mutagensis to see if that site is critical for protein stability or activity.
To address the next question of what are other proteins SIRT6 could be interacting with inside the cell. One great way for a broad look of different associating proteins would be to use the TAP tag method coupled with mass spec to identify the proteins. Once this is done to check to see if these interactions are actually true one could do co-immunoprecipitation to see if SIRT6 actually interacts with the possible proteins you found. One protein I found through my search that was shown to interact with SIRT6 was ELF5, this interaction was seen in a yeast two hybrid screen, so I would just validate this interaction with a co-immunoprecipitation.
Then to understand my final question of what other substrates SIRT6 deacetylates will help to bring a better understanding its role inside the cell by how if controls other proteins through deacetylation. A very inovative way to address this question would be to to use both a SIRT6 knockout and SIRT6 overexpressing mouse. And run a mass spec isotopical labeled analysis to look for different protein acetylation of proteins. You could then take some of the hits you acquired form that and run co-ips to look and see if SIRT6 assocated with them. And could also in vitro acetylate those proteins and then add SIRT6 to see if it deacetylates them.
In conclusion I believe that if this level of understand of SIRT6 is known it would bring a better understanding of SIRT6 role in aging and possible other key biological functions.
To address my first question about how SIRT6 is controlled post translationaly would be to look at if and how SIRT6 is modified. Such as phosophorlation, sumolyation, and ubiqutinated, all or any of these could all SIRT6 to become more active or less or even for SIRT6 to be degraded. The first possible way I would look for SIRT6 to be modified would to be to just in vitro see if SIRT6 can be modified first. This could be done simply by incubating SIRT6 with a kinase or E1/E2 sumo enzymes and then simply run a western blot and probe for these possible modifications. But to see if SIRT6 is modified in vivio, would to be use cells that where serum starved (done to activate SIRT6) and normal cell and run a mass spec analysis looking for in vivio modification and there possible location. You could also use those same cells and run a simple western to see if SIRT6 is even modified. Once you did have a possible site of modification you could then due site directed mutagensis to see if that site is critical for protein stability or activity.
To address the next question of what are other proteins SIRT6 could be interacting with inside the cell. One great way for a broad look of different associating proteins would be to use the TAP tag method coupled with mass spec to identify the proteins. Once this is done to check to see if these interactions are actually true one could do co-immunoprecipitation to see if SIRT6 actually interacts with the possible proteins you found. One protein I found through my search that was shown to interact with SIRT6 was ELF5, this interaction was seen in a yeast two hybrid screen, so I would just validate this interaction with a co-immunoprecipitation.
Then to understand my final question of what other substrates SIRT6 deacetylates will help to bring a better understanding its role inside the cell by how if controls other proteins through deacetylation. A very inovative way to address this question would be to to use both a SIRT6 knockout and SIRT6 overexpressing mouse. And run a mass spec isotopical labeled analysis to look for different protein acetylation of proteins. You could then take some of the hits you acquired form that and run co-ips to look and see if SIRT6 assocated with them. And could also in vitro acetylate those proteins and then add SIRT6 to see if it deacetylates them.
In conclusion I believe that if this level of understand of SIRT6 is known it would bring a better understanding of SIRT6 role in aging and possible other key biological functions.
Physiological understand
To get a better grasp of SIRT6 physiological role in the body I think mice would be the best model organism in part because they aging much the same ways as humans. Some very interesting mice I think that could be used would be for one a SIRT6 overexpressing mice. These mice could be compared to CR mice and also the SIRT6 knockout mice you could also see if overexpressing of SIRT6 leads to a longer life span in these mice. Also to get a better understanding of SIRT6 role later in life you could make a conditional knockout mouse such that you could knockout SIRT6 later in life to see how it may function differently in age. Another critical way to understand how SIRT6 functions over time, underdifferent conditons and in different tissues would be to due alot more microarry analysis of SIRT6 expression. This would show what tissues that SIRT6 is more prevenlent in like brain or muscle and also you could see how overtime SIRT6 levels change. With all these experiments considered I think that the physiologial understanding of SIRT6 would greatly increase and possible show that SIRT6 is a critical player in the aging process and this would draw intreset into chemical activators of SIRT6.
The search for a chemical fountain of youth?
Will SIRT6 activity hasn't yet been shown to increase a organisms life span it most likely does due to the fact that its activty is increased in CR, which increase an organisms life span. So it will be interesting to see if chemical activators of SIRT6 actually make the animals healthier and actually live longer. And how these possible activators may effect different tissues more then other due to SIRT6 being a more important player. Only time will tell when chemical libaries are used to find spicific activators of SIRT6 and how they will affect an organisms health.
